Vitamin K Epoxide Recycling Polypeptide VKORC1, a Therapeutic Target of Coumarin and Their Derivatives

ABSTRACT

The invention relates to a novel polypeptide vitamin K epoxide recycling polypeptide (VKORC1) as a target for coumarin and its derivatives. The invention further provides methods for identifying coumarin derivatives, and also claims VKORC1 polypeptides and VKORC1 nucleic acids containing a sequence abnormality associated with a VKORC1 associated deficiency such as warfarin resistance, wherein the VKORC1 polypeptides and VKORC1 nucleic acids can be used for diagnosing these deficiencies. Moreover, the invention relates to methods for identifying coumarin derivatives usable in pest control of rodents.

FIELD OF THE INVENTION

The invention relates to a novel polypeptide vitamin K epoxide recyclingpolypeptide (VKORC1) as a target for coumarin and its derivatives. Theinvention further provides methods for identifying coumarin derivatives,and also claims VKORC1 polypeptides and VKORC1 nucleic acids containinga sequence abnormality associated with aVKORC1 associated deficiencysuch as warfarin to resistance, wherein the VKORC1 polypeptides andVKORC1 nucleic acids can be used for diagnosing these deficiencies.Moreover, the invention relates to methods for identifying coumarinderivatives usable in pest control of rodents.

BACKGROUND OF THE INVENTION

Repression of untimely blood coagulation is the therapeutic option ofchoice for acute treatment and long-term prevention of thrombolicevents. Among the anti-coagulants coumarins are widely used for theprevention of thrombosis such as in patients immobilized after surgery,patients having a chronic heart failure, patients having atheroscleroticvascular disease, patients having a malignancy, and patients that arepregnant. Moreover, coumarins are the most widely used oralanticoagulants for the treatment and prophylaxis of thrombosis [Suttie,1987]. Coumarins are typically derivatives of 6-hydroxycoumarin, such as3-(acetonylbenzyl)-4-hydroxycoumarin (COUMADIN®).

The coumarins target the blood coagulation cascade indirectly byinhibition of the vitamin K cycle.

Vitamin K is an essential cofactor for the post-translational activationby gamma-carboxylation of a group of regulatory proteins, theGla-proteins. In several metabolic pathways, some key proteins requirecarboxylation for proper function. The blood coagulation cascade is thebest-studied example. Here, the procoagulant factors II, VII, IX and X,and the anticoagulant factors protein C, protein S, protein Z aredependent on gamma-carboxylation. This post-translational modificationenables the attachment of the modified proteins—in the presence ofcalcium—to phospholipid-bilayer membranes which is an essential step inthe activation of blood coagulation [Sperling et al., 1978][Esmon etal., 1975]. Other proteins requiring gamma-carboxylation are the matrixgla protein and osteocalcin, both regulators of bone metabolism [Price,1988] and the “growth arrest specific gene”, a signal transductionprotein of the cell cycle [Manfioletti et al., 1993][Stitt et al.,1995].

During gamma-carboxylation, a carboxyl group is introduced intoglutamate residues of the target proteins by the enzyme gamma-glutamylcarboxylase (GGCX) in liver microsomes [Furie & Furie, 1988][Suttie,1987]. The reaction requires as a cofactor stoichiometric amounts ofreduced vitamin. K1 hydroquinone (vitamin K1H2) which is oxidized tovitamin K-2,3 epoxide [Cain et al., 1997]. The regeneration of theactive cofactor is mediated by a multi-protein complex termed vitaminK-2,3-epoxide reductase (VKOR) [Wallin & Martin, 1985]. The same complexis targeted by the coumarin-type poisons used in rodent pest control.This “vitamin K cycle” has been characterized biochemically in greatdetail but the molecular components have not yet been purified tohomogeneity [Guenthner et al., 19981]. Moreover, the molecular nature ofcoumarin activity and the molecules interacting with coumarins are stillelusive.

It is generally appreciated in the art that although largely effective,there are a number of limitations to the use of coumarins. First of all;there are humans that are inert to coumarin treatment. The term warfarinresistance (WR) is used for individuals who maintain normal clottingfactor activities despite oral anticoagulation by coumarins (OMIMAccess. No. 122700). Autosomal dominant transmission has been observedin several pedigrees [O'Reilly et al., 1964][O'Reilly, 1970]. Combineddeficiency of all vitamin K dependent clotting factors (VKCFD) is a veryrare bleeding disorder in humans of autosomal recessive inheritance with14 cases described as yet [McMillan & Roberts, 1966][Fischer,1966][Johnson et al., 1980][Goldsmith et al., 1982][Vicente et al.,1984][Ekelund et al., 1986][Pauli et al., 1987][Leonard, 1988][Pechlaneret al., 1992][Boneh & Bar-Ziv, 1996][Brenner et al., 1998][Spronk etal., 2000][Oldenburg et al., 2000]. Clinical symptoms of the diseaseinclude episodes of perinatal intracerebral hemorrhage sometimes withfatal outcome. The bleeding tendency is usually completely reversed byoral administration of vitamin K. Additional symptoms in newborns canresemble warfarin embryopathy with nasal and distal phalangealhypoplasia and premature calcification of epiphyses [Pauli et al.,1987]. The disease may result either from a defectiveresorption/transport of vitamin K to the liver [Prentice, 1985] or frommutations in one of the genes involved in gamma-carboxylation. Insubtype 1 (VKCFD1, OMIM # 277450), mutations in the GGCX gene onchromosome 2p12 result in insufficient carboxylation of clotting factors[Brenner et al., 1998][Spronk et al., 2000]. There has been described alinkage of two kindreds with familial multiple coagulation factordeficiency (FMFD, is now re-named: VKCFD2, OMIM # 607473) to a 20 Mbinterval of the pericentric region of chromosome 16p12-q21 [Fregin etal., 2002]. Patients with VKCFD2 showed significantly increased serumlevels of vitamin K epoxide, thus suggesting a defect in one of thesubunits of the VKOR complex. Taken together, there is evidence thatthere are patients that display warfarin resistance. As a result, thereis a need to identify novel coumarins derivatives that are effectiveanticoagulants for treating these patients, and methods for identifyingthese coumarin derivatives.

The use of coumarins is associated with a risk of spontaneous bleedings,with a significant mortality rate. Moreover, the prediction of theaccurate coumarin maintenance dose is difficult. In the absence of thetarget molecule which coumarin exerts an effect on, the treatmentregimen has to be established, on a patient-by-patient basis. During thetime the optimum regimen is yet not established the patient eithersuffers from an increased risk of thrombogenesis or of an increased riskof bleeding. Therefore there is a need for a method of determining theoptimal treatment regimen that is faster and saver. Further,establishing an optimal treatment regimen is complicated by the fact,that there is a considerable delay between the administration ofcoumarins and the onset of its anticoagulant activity. Given the delayedaction of coumarin and given the fact that coumarin tends to accumulatein time there is a need for coumarin derivatives that effect bloodcoagulation faster than the coumarins known in the art. By the sametoken there is also a need for coumarins that are metabolized morerapidly so that accumulation of coumarin may be prevented or amelioratedand as a result the danger of overdosing is decreased or abolished.

It is well appreciated that if coumarin treatment is initiated during athrombic state, the levels of protein C and S decline, thus temporarilycreating a thrombogenic potential which is usually compensated for byoverlapping heparin and coumarin administration for a number of days.Again, there is a need to identify the molecular target of coumarinaction in order to be able to screen for novel coumarin derivatives thatdo not possess these limitations or at least to a lesser extend.

A coumarin therapy sometimes induces skin necrosis in patients and ifapplied during pregnancy may cause embryopathy creating a need for novelcoumarin derivatives which do not cause these effects.

There are a number of interactions between drugs and coumarins. Some ofthese drugs like Phenobarbital induce lower plasma levels of coumarinsdue to an increased metabolization of coumarin which is believed to becaused by the mixed-function oxidases like the cytochrome P450mixed-function oxidases. Such interaction is of clinical relevance ifthe appropriate regimen of e.g. Phenobarbital and coumarin has beendetermined and later on only administration of Phenobarbital isdiscontinued leading to a rise of the plasma level of coumarin whichcausing excessive anticoagulation. Other drugs like Amiodarone cause adelayed metabolization of coumarin leading again to excessiveanticoagulation if co-administered with coumarins. Since the moleculesaffected by coumarins are not known in the art there is a need todevelop novel coumarins and tools to identify the latter in order tosolve these problems.

Finally, coumarins, especially warfarin, are not only used in humans butsince the 1950s, coumarins have been in use as an active ingredient inrodenticidal compositions. The basis for the effectiveness of warfarinas a rodenticide lies in the fact that it is an effective anti-coagulantin small, multiple doses. One or two doses of the compound are seldomfatal if taken at the recommended concentration; thus the hazard ofacute toxicity to man, domestic animals, and wildlife is greatlyreduced. Usually the rodents begin to die after four or five daily dosesof the materials, and the population is greatly reduced or eradicated inapproximately three weeks. Death is caused by hemorrhages, brought aboutby the action of the warfarin in reducing the clotting power of theblood. These hemorrhages may be external or internal and can beinitiated by very slight injury or capillary damage. One of the otheradvantages of coumarins is that, because multiple ingestions arerequired to kill the rodents, they do not develop bait shyness.Beginning in 1969, rodents—particularly rats and, to a somewhat lesserextent, mice—began showing resistance to warfarin baits. The generalassumption was that such resistance had a genetic basis. As for themechanism, it is the VKORC1 complex mentioned above that is targeted byderivatives of warfarin in use for rodent pest control [Jackson et al.,1988]. Resistance to coumarin derivatives has arisen spontaneously inseveral wild rodent populations rendering the use of these drugs locallyineffective for pest control. Autosomal dominant loci for warfarinresistance have been mapped in the mouse (War) to chromosome 7 [Wallaceet al., 1976] and in rat (Rw) to the long arm of chromosome 1 [Greayses& Ayres, 1967][Kohn & Pelz, 1999]. Since the VKOR complex is the targetof the coumarin drugs resistance is thought to be mediated byalterations in one of its protein components [Jackson, 1988]. Thedevelopment of resistance in rodents has created a need for identifyingthe target of coumarins action which would facilitate the development ofnovel coumarin-derivatives for use in pest control.

Taken together it is an object of the present invention to provide atarget molecule for coumarin and its derivatives in mammals. It isanother object of the present invention to provide methods foridentifying novel coumarins which solve at least one of the problemsmentioned above. It is a further object of the present invention toidentify polypeptides and nucleic acids coding for them which causewarfarin resistance in human and non-human mammals, preferably rodents.It is also an object of the present invention to diagnose, preventand/or treat disorders and diseases selected from diseases from warfarinresistance, familial multiple factor deficiency, a disorder or diseaseassociated with increased blood coagulation such as patients sufferingfrom a thrombus and/or patients having an increased risk of developing athrombus, such as an inherited increased risk of thrombogenesis,preferably an increased risk of thrombogenesis due to a surgery or dueto pregnancy, and increased vascular calcification. Moreover, it is alsoan object of the present invention to diagnose, prevent and/or treatdiseases or disorders associated with attenuated blood coagulation, suchas hemophilia, disorder associated decreased vascular calcification anddisorders and diseases with an increased risk of bleeding. Finally, itis an object of the present invention to provide a method foridentifying coumarin and its derivatives which are effective in pestcontrol of non-human mammal and compositions for killing rodents.

SUMMARY OF THE INVENTION

In solving the above objects a vitamin K epoxide recycling polypeptide(VKORC1) is provided, comprising or consisting of a polypeptide sequenceselected from the group consisting of:

-   (a) a polypeptide sequence selected from the group consisting of a    sequence according to SEQ ID No. 1, 12, 17, 21, 25, and 27;-   (b) a polypeptide sequence of an allele of the polypeptide sequence    defined in (a);-   (c) a polypeptide sequence having at least 80% homology with the    polypeptide sequence defined in (a) or (b), which polypeptide    sequence has VKORC1 activity; and-   (d) a polypeptide sequence of a fragment of the polypeptide sequence    defined in (a), (b) or (c) having VKORC1 activity.

Moreover, according to another aspect of the present invention there isprovided a nucleic acid coding for the VKORC1 polypeptide according tothe invention (VKORC1 nucleic acid).

In addition, according to another aspect of the present invention thereis provided a method of identifying a coumarin derivative which exertsan effect onto the activity of VKORC1 polypeptide according to theinvention comprising the steps of:

-   (I) providing a host cell having been introduced the VKORC1 nucleic    acid or a vector containing the VKORC1 nucleic acid;-   (II) expressing the VKORC1 polypeptide in the host cell;-   (III) administering a candidate coumarin derivative;-   (IV) determining the activity of VKORC1 polypeptide (candidate    activity value);-   (V) comparing the candidate activity value with a control activity    value; and-   (VI) identifying the candidate coumarin derivative as a coumarin    derivative exerting an effect onto the activity of the VKORC1    polypeptide, provided the candidate activity value is significantly    different from the control activity value.

Furthermore, according to another aspect of the present invention thereis provided a method of determining a VKORC1 polypeptide sequence whichconveys a coumarin effect exerted onto VKORC1 activity, comprising thesteps of:

-   (I) providing a cell expressing the VKORC1 polypeptide according to    the invention, which VKORC1 polypeptide has at least one sequence    abnormality;-   (II) administering coumarin or a derivative thereof to the cell;-   (III) determining the activity of the VKORC1 polypeptide (sequence    abnormality activity value); and-   (IV) comparing the sequence abnormality activity value with the    control sequence activity value,    wherein a significant deviation of the sequence abnormality activity    value from to the control sequence activity value is indicative that    the sequence abnormality of the VKORC1 polypeptide conveys the    coumarin effect exerted onto VKORC1 polypeptide.

In another aspect of the present invention there is provided a VKORC1polypeptide according to the invention, wherein the VKORC1 polypeptidecontains at least one sequence abnormality, which exerts an effect onthe activity of the VKORC1 polypeptide.

Moreover, according to another aspect of the present invention there isprovided a method of diagnosing a VKORC1 associated deficiency in apatient comprising the steps of:

-   (I) amplifying a DNA sample obtained from the patient or reverse    transcribing a RNA sample obtained from the patient into a DNA and    amplifying the DNA; and-   (II) analyzing the amplified DNA of step (I) to determine at least    one sequence abnormality in a nucleic acid sequence coding for the    VKORC1 polypeptide of claim 1 or in an amino acid sequence of the    VKORC1 polypeptide;    wherein the determined sequence abnormality is indicative of the    patient suffering from a VKORC1 associated deficiency; preferably    the sequence abnormality exerts an effect on the activity of the    VKORC1 polypeptide.

In addition, according to another aspect of the present invention thereis provided a method of identifying a coumarin derivative which istoxicologically effective in warfarin-resistant rodents comprising thesteps of:

-   (I) providing a warfarin-resistant rodent;-   (II) administering a candidate coumarin derivative to the rodent;-   (III) determining the toxicity of the candidate coumarin derivative    onto the rodent (candidate coumarin derivative toxicity value);-   (IV) comparing the candidate coumarin derivative toxicity value with    a control coumarin toxicity value;-   (V) identifying the candidate coumarin derivative as a    rodenticidally effective coumarin derivative provided that the    candidate coumarin derivative toxicity value is significantly larger    than the control coumarin toxicity value.

According to another aspect of the present invention, the identifiedcoumarin derivatives can be included into a composition for killingrodents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of the candidate interval of 3 cM in thegenetic map containing the VKORC1 gene locus in human, rat and mouse.The ideogram of human chromosome 16 is shown, the area of homozygosityin the families 1 and 2 extends from 16p11.2 to 16q13 corresponding toapproximately 25 Mb. In the right part of the figure there arehomologous parts of mouse and rat-chromosomes. Synthenic genes of16p11.2 and 16q12.1 with homologous counterparts in Mus musculus (MMU)and Rattus norvegicus (RNO) are depicted. The loci for phenotyperesistance to warfarin in the mouse (War) and rat (Rw) are mapped toregions homologous to 16p11.2. MMU: Mus musculus; RNO: Rattusnorvegicus; PRKCB1; Prkcb; IL4R: Interleukin4 receptor α (human); Il4ra:Interleukin4 receptor α (murin); Il4r: Interleukin4 receptor α (rat);SPS2: Selenophosphate synthetase (human); Sps2: Selenophosphatesynthetase (murin/rat); HUMMLC2B: Myosin light Chain2 (human); Mylpf:Myosin light chain 2 (murin); Myl2: Myosin light chaun 2 (rat); SPN:Sialophorine (human); Spn: Sialophorine (murin/rat).

FIG. 2 displays VKORC1 mutations in human vitamin K dependent clottingfactor 2 (VKCFD2) and warfarin resistance (WR) patients. The upper partof the figure shows the segregation of the R98W mutation in two VKCFD2families and the electropherograms of a homozygous mutant (left)compared to a control (right). The bottom part of the figure shows theheterozygous mutations of four WR patients. (85G>T, 134T>C, 172A>G,383T>G) and a Rw rat (416A>G).

FIG. 3 shows a sequence alignment of VKORC1 and VKORC1 like protein 1(VKORC1L1) polypeptides. The alignment was generated with CLUSTALW andPRETTYBOX. Human (hVKORC1), mouse (mVKORC1) and rat VKORC1 (rVKORC1) andVKORC1L1 polypeptides, i.e. VKORC1L1 of human (hVKORC1L1), mouse(mVKORC1L1) and Fugu rubripes (fVKORC1L1), share approximately 84%sequence identity within both groups and approximately 50% identitybetween both groups of proteins. xVKORC1 depicts the VKORC1 polypeptidesequence of Xenopus laevis, fVKORC1 the VKORC1 polypeptide sequence ofFugu rubripes, and aVKORC1 the VKORC1 polypeptide sequence of Anophelesgambiae. Tree analysis allows grouping the Fugu rubripes, Xenopus laevisand Anopheles gambiae proteins to the appropriate group. The locationsof the predicted transmembrane domains are underlined. Residues 29, 45,58 and 128 mutated in WR patients are conserved in all species. Thearginine at position 98 mutated in the VKCFD2 patients is conserved inhuman, rat and mouse (plus sign).

FIG. 4 displays a northern blot analysis of VKORC1 in fetal and adulthuman tissues. The upper blot depicts a northern blot of adult tissue,whereas the lower blot to depicts a northern blot of fetal tissue. Formore details see Example 4. The lines with fragments of the sizes 2.4,4.4, 7.5, UND 9.5 KB indicate molecular weight markers and allowestimation of the size of the all visible bands)

FIG. 5 shows the subcellular location of VKORC1. For more details seeexample 6. To this end, COS-7 cells transiently transfected with VKORC1constructs were stained with anti-calnexin (red; left column) andanti-GFP or anti-myc, respectively (green; middle column). Mergedfigures of the double-stained cells are shown in the right column. BothVKORC1 constructs (tagged with GFP or myc) co-localize with the ERspecific calnexin staining. The control construct (pEGFP-N1) shows adiffuse staining pattern throughout the cytoplasm.

FIG. 6 displays a list of siRNA sequences for homo sapiens VKORC1 andprimers endoding these siRNAs which can be used to express them usingfor example the siLentGene™ U6 Cassette RNA Interference System.

FIG. 7 displays locations of siRNA targets in the coding sequence ofhomo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1),which are shown in light grey; regions which are part of two possiblesiRNA targets are shown in darker grey; and regions with two or morepossible siRNA sequences are shown in an even darker grey.

FIG. 8 provides a list of PCR primer sequences and PCR conditions foramplification of Homo sapiens VKORC1 and Homo sapiens VKORC1L1.

FIG. 9 provides a listing of the sequences their respective SEQ ID NOs.

FIG. 10 shows VKOR activities of HEK293 cells transfected with VKORC1cDNA. Values are given as percent vitamin K epoxide converted intovitamin K quinone (product/residual substrate+product). Wildtype VKORC1activity is also defined by being sensitive to warfarin (4.3% residualactivity at 80 μM warfarin compared to not inhibited). Mutations Y139Cand V29L leading to resistance to warfarin exhibit 69 and 11% residualactivity at 80 μM warfarin respectively). All tests were run induplicate. Untransfected and mock-transfected showed 1.49 and 0.96%activities, and were >90% inhibited by 10 μM warfarin. For furtherdetails see Example 7.

FIG. 11 shows the amino acid sequence of Homo sapiens vitamin K epoxiderecycling polypeptide (HS_VKORC1; SEQ ID NO: 1)

FIG. 12 shows the nucleic acid coding sequence of Homo sapiens vitamin Kepoxide recycling polypeptide (HS_VKORC1; SEQ ID NO: 2)

FIG. 13 shows the result of an ARMS-PCR experiment to determine whetheror not a tested rat is warfarin resistant. Wildtype rats exhibited aband at 123 bp (probe# 3351, 3133, 3137, 3142, 4724, 4684, 3138, 3162),rats homozygous to the mutation (probe# 4701) exhibited a band at 101 bpand finally, rats with the heterozygous mutation (probe# 3066, 3350,3352, 3354, 3139, 3140, 4754, 3146, 3148, 3149) showed two bands, one at101 and another band at 123 bp. For further details see Example 9.

DETAILED DESCRIPTION OF THE INVENTION

In order to meet the needs for developing novel coumarin derivatives andfor identifying the target of coumarin and its derivatives the vitamin Kepoxide recycling polypeptide (VKORC1) was cloned. This gene waspreviously unknown spanning a genomic region of 5126 bp and comprisingthree exons coding for a protein of 163 amino acids. Topology analysissuggests a highly hydrophobic protein with at least two transmembranedomains. This is compatible with the known location of the VKORC1complex activity in ER membranes and with immunofluorescence data inCOS-7 cells transfected with VKORC1 constructs (FIG. 5).

The VKORC1 gene was surprisingly identified in a mutant screen ofwarfarin resistant patients (for details cf. examples 1 and 2).According to the present invention, there has been surprisinglyidentified a gene, VKORC1, which is mutated in patients with a combineddeficiency of all vitamin K dependent coagulation factors (VKCFD2) andwith warfarin resistance (WR), respectively, showing that VKORC1polypeptide contains a binding site for warfarin and is a target ofcoumarin and its derivatives. The evidence that the mutations arecausative of the two phenotypes is as follows:

-   (i) an R98W mutation segregates with the disease in two unrelated    families with VKCFD2;-   (ii) this arginine at position 98 is conserved in the human and in    the homologous mouse and rat genes, respectively;-   (iii) three warfarin resistant brothers share an R58G substitution;-   (iv) this amino acid and the other residues found mutated in two    more unrelated WR patients (V29L and L128R) are conserved in all    species analyzed except for three bacterial genes (see FIG. 3); and-   (v) none of the 5 presumed mutations was found in 192 control DNA    samples.

Moreover, homology searches in genome and protein databases have notrevealed any similarities of VKORC1 to any protein or peptide domain ofannotated function. However, homologous genes are found in vertebrates(rat, mouse, Xenopus, Fugu), insects (Anopheles) and bacteria (FIG. 3).Surprisingly, the three mammals and Fugu each have a second VKORC1-likegene of moderate similarity to the cognate gene. A number of amino acidpositions within these genes are conserved throughout evolution. This isin accordance with the well established fact thatgamma-carboxylation—and thus the use of vitamin K as a cofactor of thisprocess—is an evolutionary old post-translational protein modification[Bandyopadhyay et al., 2002].

A substitution of valine 29, arginine 58 leucine 128—although dispersedover the entire VKORC1 polypeptide—obviously renders the inhibition ofVKORC1 activity by warfarin ineffective. It can be speculated that theseamino acids functionally co-operate in the tertiary structure of theVKORC1 protein 1. Taken together the mutation data in patients with twodifferent phenotypes provide VKORC1 as the target protein, both forvitamin K and warfarin binding.

In one aspect of the present invention there is provided a vitamin Kepoxide recycling polypeptide (VKORC1) comprising, preferably consistingof, a polypeptide sequence selected from the group consisting of:

-   (a) a polypeptide sequence selected from the group consisting of a    sequence according to SEQ ID No. 1, 12, 17, 21, 25, and 27;-   (b) a polypeptide sequence of an allele of the polypeptide sequence    defined in (a);-   (c) a polypeptide sequence having at least 80% homology with the    polypeptide sequence defined in (a) or (b), which polypeptide    sequence has VKORC1 activity; and-   (d) a polypeptide sequence of a fragment of the polypeptide sequence    defined in (a), (b) or (c) having VKORC1 activity.

Preferably, the VKORC1 polypeptide is a target for coumarin and itsderivatives in mammals.

Within the meaning of the invention the term “VKORC1 polypeptide” refersto the full length sequence of the VKORC1 polypeptide as defined in thepreceding paragraph. The term “VKORC1 polypeptide” also encompassesisolated VKORC1 polypeptides and VKORC1 polypeptides that are preparedby recombinant methods, e.g. by isolation and purification from asample, from a host cell expressing the VKORC1 polypeptide, by screeninga library and by protein synthesis, all of these methods being generallyknown to the person skilled in the art. Preferably, the entire VKORC1polypeptide or parts thereof can be synthesized, for example, with theaid of the conventional synthesis such as the Merrifield technique. Morepreferably, the term “VKORC1 polypeptide” also encompasses polypeptideswhich have, a sequence homology of about 80%, preferably about 90%, inparticular about 95%, especially about 98% with the VKORC1 polypeptideaccording to one of SEQ ID No. 1, 12, 17, 21, 25, and 27, provided thatsuch VKORC1 polypeptide has VKORC1 activity. Moreover, it is preferredthat the term “VKORC1 polypeptide” also encompasses homologouspolypeptides which originate from organisms other than human, preferablyfrom non-human mammals such as, rodents, e.g. mouse, rats, or monkeysand pigs and other vertebrates and invertebrates, such as those aminoacid sequences according to SEQ ID Nos. 12, 17, 21, 25, 27, providedthat such VKORC1 polypeptide has VKORC1 activity. It is even morepreferred that the term “VKORC1 polypeptide” also includes VKORC1polypeptides which are encoded by different alleles of the gene, indifferent individuals, in different organs of an organism or indifferent developmental phases, provided that such VKORC1 polypeptidehas VKORC1 activity. It is further intended that the term “VKORC1polypeptide” preferably also encompasses naturally occurring orsynthetic mutations that exert no or only insignificant effects onto theactivity of the VKORC1 polypeptide. Other polypeptides preferablyencompassed by the term “VKORC1 polypeptide” include VKORC1 polypeptidesthat may arise from differential splicing of the VKORC1 transcript,provided that such VKORC1 polypeptide has VKORC1 activity.

The term “fragment of the polypeptide sequence” is intended to encompasspartial sequences of VKORC1 polypeptides, which fragments comprise,preferably consist of at least about 60%, preferably at least about 70%,more preferably at least about 80%, more preferably at least about 90%,even more preferably at least about 95% of the full length sequence ofthe VKORC1 polypeptide. In particular, it is preferred that the fragmentconsists of a single contiguous sequence of the VKORC1 polypeptide butit may also contain at least two, at least three or at least about fivedifferent sequence portions of a VKORC1 polypeptide according to theinvention which may or may not be interspaced by a heterologeoussequence or contain no extra polypeptide sequence at all.

The term “sequence homology” is understood as the degree of identity (%identity) of two sequences, that in the case of polypeptides can bedetermined by means of for example BlastP 2.0.1 and in the case ofnucleic acids by means of for example BLASTN 2.014, wherein the Filteris set off and BLOSUM is 62 (Altschul et al., 1997).

“VKORC1 activity” within the meaning of the present invention isintended to mean the biological activity of the VKORC1 polypeptide ofSEQ ID No. 1. More preferably, “VKORC1 activity” is defined as theactivity of the VKORC1 polypeptide to enzymatically convert (or supportthe enzymatic conversion) of vitamin K2,3-epoxide to vitamin K-quinoneand/or the conversion of vitamin k quinone to vitamin K hydroquinone.VKORC1 activity may be determined using an assay based on theexperiments described in detail in example 7 and FIG. 10. Using thatassay, a measured percentage of vitamin K epoxide converted into vitaminK quinone (product/substrate+product) in cells expressing a given VKORC1polypeptide, or a nucleic acid molecule coding for such VKORC1polypeptide, which raises the basal VKOR-activity of HEK293 cells fromabout 1% (1.49 and 0.96% for untransfected and mock-transfected HEK293cells, respectively) to about 15% or more, preferably to about 18% ormore, preferably to about 20% or more, most preferably to about 25% ormore is considered a VKORC1 activity within the meaning of theinvention.

The VKORC1 polypeptides according to the present invention may beproduced by a method described in more detail below. Among others, theVKORC1 polypeptides are useful for identifying coumarin derivatives thatavoid the problems described above. In particular they are useful foridentifying coumarin derivatives, that effectively inhibit VKORC1activity and that in independent assays are tested for (1) theirmetabolic half life in order to identify coumarin derivatives that aremetabolized faster than the coumarins known in the art, (2) theirability to cause skin necrosis to identify coumarin derivatives that donot cause skin necrosis or to a lesser extend than the coumarins knownin the art, (3) coumarin derivative-drug interactions in order toidentify coumarin derivatives with lesser side effects than thecoumarins known in the art. Moreover, the VKORC1 polypeptides accordingto the present invention are useful for identifying a VKORC1 sequenceinteracting with coumarin and its derivatives, and for treating patientshaving a decreased or increased VKORC1 activity relative to controllevels.

In another aspect the present invention relates to a VKORC1 nucleic acidcomprising, preferably consisting essentially of a nucleic acid sequenceselected from the group consisting of:

-   (a) a nucleic acid sequence coding for the VKORC1 polypeptide    according to the invention;-   (b) a nucleic acid sequence selected from the group consisting of a    sequence according to SEQ ID No. 2, 13, 18, 22, 26, and 28;-   (c) a nucleic acid sequence which hybridizes under stringent    conditions to the nucleic acid sequence defined in (a) or (b), which    nucleic acid sequence codes for a polypeptide having VKORC1    activity;-   (d) a nucleic acid sequence which, but for the degeneracy of the    genetic code, would hybridize, preferably under stringent    conditions, to the nucleic acid defined in (a), (b) or (c) and which    nucleic acid sequence codes for a polypeptide having VKORC1    activity; and-   (e) a fragment of the nucleic acid sequence defined in (a), (b), (c)    or (d), which fragment codes for a polypeptide having VKORC1    activity.

Preferably, the VKORC1 nucleic acid is a target for coumarin and itsderivatives in mammals.

The term “VKORC1 nucleic acid” relates to RNA or DNA, which may be asingle or preferably a double stranded molecule. The sequence of theVKORC1 nucleic acid may further comprise at least one intron and/or onepolyA sequence. The term “VKORC1 nucleic acid” may also encompass aprecursor stage, for example a propolypeptide or prepropolypeptide,thereof. It is also understood that untranslated sequences can bepresent at the 5′ end and/or the 3′ end of the nucleic acid, without theactivity of the encoded polypeptide being significantly altered.However, the DNA region encoding the VKORC1 polypeptide is particularlypreferred. In eukaryotes, this region begins with the first start codon(ATG) which is located in a Kozak sequence (Kozak, 1987) and extends tothe next stop codon (TAG, TGA or TAA) which is located in the samereading frame as the ATG. In the case of prokaryotes, this region beginswith the first AUG (or GUG) after a Shine-Dalgarno sequence and endswith the next stop codon (TAG, TGA or TAA) which is located in the samereading frame as the ATG. Moreover, the term “VKORC1 nucleic acid” mayalso encompass sequences which exhibit at least about 70%, in particularat least about 80%, especially at least about 90%, sequence homologywith the sequence according to SEQ ID No. 2, 13, 18, 22, 26, and 28,preferably to the sequence according to SEQ ID No. 2, provided that theVKORC1 polypeptide encoded by such is nucleic acid has VKORC1 activity.In a preferred embodiment of the invention the nucleic acid comprises anucleic acid having a sequence complementary and/or antisense to aVKORC1 nucleic acid as defined in the preceding paragraph. The VKORC1nucleic acid may also comprises a non-functional mutant variant of theVKORC1 nucleic acid as defined above, such a variant containing a singlenucleotide polymorphism (SNP) such as the nucleic acid sequencesaccording to SEQ ID No. 8 and 9, provided that the VKORC1 polypeptideencoded by such nucleic acid has VKORC1 activity.

The term “stringent hybridization conditions” is to be understood, inparticular, as meaning those conditions in which a hybridization takesplace, for example, at 60° C. in 2.5×SSC buffer followed by severalwashing steps at 37° C. in a lower buffer concentration and remainsstable.

The term “fragment of the nucleic acid sequence coding for a polypeptidehaving VKORC1 activity” is understood to encompass nucleic acid sequencefragments comprising, preferably consisting of at least about 60%,preferably at least about 70%, more preferably at least about 80%, morepreferably at least about 90%, even more preferably at least about 95%of the full length sequence coding for VKORC1 polypeptide according tothe invention, preferably coding for the polypeptide according to SEQ IDNo. 1, provided that the polypeptide encoded by such fragment has VKORC1activity. In particular, it is preferred that the fragment consists of asingle contiguous sequence coding for the VKORC1 polypeptide but it mayalso contain at least two, at least three or at least about fivedifferent sequence portions, which may or may not be interspaced by aheterologeous sequence or contain no extra nucleic acid sequence at all,provided that all the sequence portions are arranged in the same readingframe. It is essential to the definition of these fragments that theydisplay VKORC1 activity.

The VKORC1 nucleic acids can be produced by methods generally known tothe skilled artisan. Nucleic acids may be prepared synthetically. Thus,the VKORC1 nucleic acids can, for example, be synthesized chemically,e.g. according to the phosphotriester method, with the aid of the DNAsequences as defined above and/or with the aid of the polypeptidesequences which are likewise defined above such as the SEQ ID No. 1 andby referring to the genetic code (see, e.g., Uhlmann, & Peyman, 1990).Preferably the VKORC1 nucleic acids are produced by recombinant genetechnology methods generally known to the person skilled in the art.

Among others, the VKORC1 nucleic acids are useful (1) for identifyingcoumarin derivatives that avoid the problems described above, (2) forproducing PCR primers, DNA and RNA probes, siRNA or shRNA, and forVKORC1 polypeptide, (3) for treating patients having a decreased orincreased VKORC1 activity relative to control values, and (4) foridentifying coumarin derivatives that may be employed for pest controlof rodents, all of which are described in detail below.

The present invention further provides in another aspect a method ofproducing a VKORC1 polypeptide, preferably a polypeptide according toSEQ ID No. 1, 12, 17, 21, 25, and 27, comprising the steps of:

-   (I) providing a host cell having been introduced the VKORC1 nucleic    acid, preferably a nucleic acid according to SEQ ID No. 2, 13, 18,    22, 26, and 28, or a vector containing the VKORC1 nucleic acid;-   (II) expressing the VKORC1 polypeptide in the host cell; and-   (III) isolating the VKORC1 polypeptide from the host cell.

The host cell can be any host cell as defined below. Methods forselecting and culturing the host cells and for causing the host cells toexpress a polypeptide are generally known to the person skilled in theart. The same is true for methods of isolating the expressed polypeptidefrom the host cell; to this end an antibody according to the inventionmay be used for immunoaffinity precipitation. As an alternative thevector may contain a (poly)peptide tag that allows immunoaffinityprecipitation by tag specific antibodies according to standard protocolsknown to the skilled worker (see also below).

In another aspect the present invention relates to a fusion proteincomprising, preferably consisting essentially of,

-   (a) the VKORC1 polypeptide as defined above, preferably according to    SEQ ID No. 1, 12, 17, 21, 25, 27 or a polypeptide encoded by the    VKORC1 nucleic acid, preferably a nucleic acid according to SEQ ID    No. 2, 13, 18, 22, 26, 28 and-   (b) a heterologeous part.

This involves fusion proteins which contain the above-described VKORC1polypeptide, with the fusion proteins themselves already being active oronly becoming active after the heterologeous part has been eliminated.To this end the heterologeous part may further comprise a peptidecleavable by a protease. The heterologeous part may be a proteinaceouscompound, a peptide or a different compound. These fusion proteinsinclude in particular, fusion proteins having a content of about 1-300,preferably about 1-200, particularly preferably about 1-150, inparticular about 1-100, especially about 1-50 foreign amino acidsconstituting the heterologeous part. The heterologeous part can belocated N-terminally, C-terminally and/or internally relative to theVKORC1 polypeptide. Examples of such peptide sequences are prokaryoticpeptide sequences which can be derived, for example, from E. coligalactosidase.

Other preferred examples of peptide sequences for fusion proteins arepeptides which facilitate detection of the fusion protein; examples ofthese are the green fluorescent protein or functional variants thereof.It is also possible to add on at least one further “polypeptide tag”e.g. for the purpose of purifying the previously described VKORC1polypeptides. For example, suitable protein tags enable the fusionproteins which are to be purified, to be absorbed with high affinity toa matrix. This is then followed, for example, by stringent washing withsuitable buffers without eluting the to fusion proteins to anysignificant extent, and, subsequently, specific elution of the fusionproteins. Examples of the protein tags which are known to the skilledperson are a (His)₆ tag, a Myc tag, a FLAG tag, a hemagglutinin tag, aglutathione transferase (GST) tag, intein having an affinitychitin-binding tag and a maltose-binding protein (MBP) tag. Theseprotein tags can be located N-terminally, C-terminally and/or internallyrelative to the VKORC1 polypeptide. Fusion proteins are for exampleuseful for the production of VKORC1 production and subsequent isolation.Moreover, the fusion proteins may be employed for detecting thelocalization of the expression product in the cell or the organism.

In another aspect the present invention relates to a vector comprising aVKORC1 nucleic acid as defined above, preferably the VKORC1 nucleic acidaccording to SEQ ID No. 2. Preferably the vector is an expressionvector. In order to enable the VKORC1 nucleic acids to be used accordingto the present invention they may be introduced into a eukaryotic orprokaryotic cell by means of transfection, transformation or infection,and thereby enable the polypeptide to be expressed. The VKORC1 nucleicacid can be present as a plasmid, or as a part of a viral or non-viralvector. Particularly suitable viral vectors in this connection are:baculoviruses, vaccinia viruses, adenoviruses, adeno-associated virusesand herpes viruses. Particularly suitable non-viral vectors are forexample: virosomes, liposomes, cationic lipids and polylysine-conjugatedDNA. The vectors can be prokaryotic or eukaryotic expression vectors.Examples of prokaryotic expression vectors are the pGEM vectors or pUCderivatives, which are used for expression in E. coli, and examples ofeukaryotic expression vectors are the vectors p426Met25 or p426GAL1(Mumberg et al., 1994) which are used for expression in Saccharomycescerevisiae, the Baculovirus vectors, as disclosed in EP B1 0 127 839 orEP B1 0 549 721, which are used for expression in insect cells, and thevectors Rc/CMV and Rc/RSV, or SV40 vectors, which are used forexpression in mammalian cells, with all these vectors being generallyavailable. In general, the expression vectors also contain promoterswhich are suitable for the respective cell, such as the trp promoter forexpression in E. coli (see, e.g., EP-B1-0 154 133), the Met 25, GAL 1 orADH2 promoter for expression in yeasts (Russel et al, 1983; Mumberg, seeabove), and the baculovirus polyhedrin promoter for expression in insectcells (see, e.g., EP B1 0 127 839).

Promoters which permit constitutive, regulatable, tissue-specific, celltype-specific, cell cycle-specific or metabolism-specific expression ineukaryotic cells are suitable, for example, for expression in mammaliancells. Regulatable elements in accordance with the present invention arepromoters, activator sequences, enhancers, silencers and/or repressorsequences. Examples of preferred regulatable elements which permitconstitutive expression in eukaryotes are promoters which are recognizedby RNA polymerase III or viral promoters, CMV enhancer, CMV promoter,SV40 promoter or LTR promoters, e.g. derived from MMTV (mouse mammarytumor virus; Lee et al., 1981) and other viral promoter and activatorsequences which are derived from, for example, HBV, HCV, HSV, HPV, EBV,HTLV or HIV. Examples of regulatable elements which permit inducibleexpression in eukaryotes are the tetracycline operator in combinationwith an appropriate repressor (Gossen et al., 1994). The expression ofVKORC1 nucleic acids preferably takes place under the control oftissue-specific promoters. The expression vectors may be used forpreparing a VKORC1 polypeptides, DNA or RNA probes, or siRNA or shRNA,which can be used in accordance with the invention.

In another preferred embodiment of the present invention the vector is aknock-out gene construct. The construction of such constructs andmethods for constructing knock-out animals are known to the personskilled in the art, for example, from the U.S. Pat. No. 5,625,122; U.S.Pat. No. 5,698,765; U.S. Pat. No. 5,583,278 and U.S. Pat. No. 5,750,825.Such vectors are for example useful for generating knock-out cells andanimals which in turn can be used to identify disorders and diseasesassociated with impaired VKORC1 activity.

“Impaired VKORC1 activity” within the meaning of the invention relatesto a level of activity and/or expression of the VKORC1 protein that isless than control level activity (as defined above) and/or expressiondetermined in a healthy subject; the respective levels of activity mayalso be determined based on the assay as described in Example 7.

In another aspect the present invention relates to a host cell,preferably a non-human embryonic stem cell, containing one of theaforementioned vectors, preferably an expression vector or a knock-outgene construct. The host cell can be any cell suitable for expression ofVKORC1 polypeptides and/or VKORC1 nucleic acids, preferably aHEK293-EBNA cell. Cells can be either prokaryotic or eukaryotic cells,heterologeous or autologous cells. Examples of prokaryotic cells are E.coli and examples of eukaryotic cells include primary hepatocytes cells,yeast cells, for example Saccharomyces cerevisiae or insect cells. Morepreferably the host cell is a cell line, e.g. a COS-cell such as COS-7cells or hepatocytes cell lines such as HepG2 cells. Moreover the hostcell is preferably a non-human embryonic stem cell. Methods forselecting and culturing host cells and for causing the host cells toexpress a polypeptide are generally known to the person skilled in theart. Processes for the transformation of cells and/or stem cells arelikewise well known to a person skilled in the art and include, forexample, electroporation or microinjection. The host cells of thepresent invention can for example be employed for methods of identifyingcoumarin derivatives, for producing VKORC1 polypeptides and VKORC1nucleic acids, siRNAs and shRNAs according to the invention, and forscreening new drugs such as coumarin derivatives effecting VKORC1activity and/or expression.

In another aspect the present invention relates to the provision of atransgenic non-human mammal containing a host cell according to theinvention, preferably a non-human embryonic stem cell, as defined above.Transgenic animals in general show a tissue-specifically increasedexpression of the VKORC1 polypeptides and/or VKORC1 nucleic acids andcan be used for the analysis of coagulation disorders and warfarinresistance and for development and evaluation of therapeutic strategiesfor such disorders. Transgenic animals may further be employed in theproduction of VKORC1 polypeptides. The polypeptide produced by theanimal may for example be enriched in a body fluid of the animal.

In another preferred embodiment of the present invention it is provideda transgenic non-human mammal which is transgenic for a VKORC1polypeptide which contains at least one sequence abnormality exerting aneffect on the activity of the VKORC1 polypeptide as defined in detailbelow. Preferably the animal is transgenic for the VKORC1 polypeptideaccording to SEQ ID No. 1, 12, 17, 21, 25, and 27, and preferably thesequence abnormality is selected from the group consisting of V29L, V45AR58G, R98W, L128R, and Y139C. These transgenic animals are for exampleuseful (1) for testing coumarin derivatives for warfarin resistance; (2)for identifying novel coumarin derivatives that are effectiveanticoagulants in organisms that are resistant or less susceptible toanticoagulant treatment with coumarins known in the art, such aswarfarin resistance patients; and (3) as a source of cells expressingVKORC1 polypeptide and/or VKORC1 nucleic acids. Moreover, these animalscan be used for identifying novel coumarin derivatives.

Methods for the preparation of transgenic animals, in particular oftransgenic mice, are likewise known to the person skilled in the artfrom DE 196 25 049 and U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,625,122;U.S. Pat. No. 5,698,765; U.S. Pat. No. 5,583,278 and U.S. Pat. No.5,750,825 and include transgenic animals which can be produced, forexample, by means of direct injection of expression vectors according tothe present invention into embryos or spermatocytes or by injection ofthe expression vectors into the pronucleus of the fertilized ovum or bymeans of the transfection of expression vectors into embryonic stemcells or by nuclear transfer into appropriate recipient cells (Polites &Pinkert, 1994; Doetschman, in Pinkert, 1994, supra; Wood in Pinkert,1994, supra; Monastersky in Pinkert, 1994, supra).

Within the meaning of the term “VKORC1 associated deficiency” isintended to encompass a disorder or disease that is associated withwarfarin resistance, i.e. the patient displays a reduced or abolishedsusceptibility to treatment with coumarin or its derivatives, preferablythe warfarin resistance results from a sequence abnormality of theVKORC1 polypeptide. Moreover, the term preferably also encompassesdisorders or diseases associated with a level of activity and/orexpression of VKORC1 that differs significantly from the condition inhealthy patients, preferably the expression of VKORC1 polypeptide and/orits activity is significantly reduced or abolished which can e.g. bedetermined by measuring the prothrombin time, e.g. by internationalnormalized ration (INR) protocol. Such VKORC1 associated deficiency maybe caused by a sequence abnormality in the VKORC1 polypeptide or VKORC1nucleic acid as described in detail below. Moreover, when the level ofexpression and/or activity of VKORC1 polypeptide or VKORC1 nucleic acidis reduced or even completely abolished, gamma-carboxylation of vitaminK dependant proteins may also be impaired as well. Thus, in this contextthe term “VKORC1 associated deficiency” also encompasses diseases and/ordisorder selected from familial multiple factor deficiency, a disorderor disease associated with decreased blood coagulation, such ashemophilia and a disorder associated decreased vascular calcification,diseases and/or disorders associated with impaired gamma-carboxylationof vitamin K dependant proteins.

It is also conceivable that the level of expression and/or activity ofVKORC1 polypeptide will be increased relative to the condition inhealthy patients. Such deficiencies which are also encompassed by theterm the “VKORC1 associated deficiency” may be caused by a sequenceabnormality in the VKORC1 polypeptide and/or the corresponding gene.Moreover, when the level of expression and/or activity of VKORC1polypeptide or VKORC1 nucleic acid is increased, gamma-carboxylation ofvitamin K dependant proteins may be facilitated as well. Thus, in thiscontext the term “VKORC1 associated deficiency” may further comprisedeficiencies selected from diseases or disorders associated withincreased blood coagulation including patients suffering from a thrombusand/or patients having an increased risk of developing a thrombus,preferably due to a sequence abnormality in the VKORC1 polypeptide orits gene, diseases and/or disorders associated with improvedgamma-carboxylation of vitamin K dependant proteins.

It is also possible that a sequence abnormality in the VKORC1polypeptide and/or the corresponding gene may increase thesusceptibility to treatment with coumarin and its derivatives in apatient having such sequence abnormality. As a result patientsundergoing coumarin treating may show very low blood coagulation values.Such disorders associated with increased susceptibility to treatmentwith coumarin are also intended to be encompassed by the term “VKORC1associated deficiency”. Patients carrying a VKORC1 gene having astop-mutation suffer from such deficiency associated with a increasedcoumarin sensitivity.

In another aspect the present invention relates to a DNA or a RNA probedirected against the VKORC1 nucleic acid according to the invention,preferably against the VKORC1 nucleic acid selected from according toSEQ ID No. 2, 13, 18, 22, 26, and 28. A probe is a nucleic acid moleculethat allows detection of a VKORC1 nucleic acid it is directed againstit. The probe has a sequence which hybridizes to the target sequence,i.e. the VKORC1 nucleic acid. Preferably the probe allows specificdetection of the VKORC1 nucleic acid, i.e. at least under stringenthybridization conditions it does not bind to molecules other than theparticular VKORC1 nucleic acid. Suitable probes are, for example, DNA orRNA fragments having a length of about 10 to about 492 nucleotides,preferably having a length of about 10 to about 400 nucleotides,preferably about 10 to about 250 nucleotides, in particular having alength of about 10 to about 150 nucleotides, in particular having thefull length of the coding sequence, which sequence can be derived fromthe VKORC1 polypeptides, preferably selected from the VKORC1 polypeptideaccording to SEQ ID No. 1, 12, 17, 21, 25, and 27 or taken directly fromthe VKORC1 nucleic acid, preferably selected from SEQ ID No. 2, 13, 18,22, 26, and 28. The probes may additionally contain a label suitable fordirect or indirect detection such as biotin, a fluorescent label such asfluorescein or a radioactive label such as [H]³ or other labels known tothe skilled worker. Detection may be carried out by methods generallyknown to the skilled worker including northern blotting and cDNA libraryblotting techniques. The construction of the probes according to thepresent invention is also known to the skilled worker (cf. constructionof nucleic acids described above). Such probes can for example be usedfor diagnostic purposes and may preferably comprise or consist of theprobes suitable for detection of a sequence abnormality such as thoseselected from the sequences according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9,14, and 94.

In another aspect the present invention relates to a PCR primer,preferably a set of at least two PCR primers directed against the VKORC1nucleic acid, preferably against the VKORC1 nucleic acid according toSEQ ID No. 2, 13, 18, 22, 26, and 28. Suitable primers are, for example,DNA fragments having a length of about 10 to about 100 nucleotides,preferably having a length of about 15 to about 50 nucleotides, inparticular having a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, preferably about 30 nucleotides. The design andsynthesis of such primers is generally known to the person skilled inthe art. The primers may additionally contain restriction sites, e.g.suitable for integration of the amplified sequence into vectors, orother adapters or overhang sequences, e.g. having a label as describedin the preceding section. For example, according to the presentinvention it is possible to prepare a diagnostic based on the polymerasechain reaction (PCR), suitable for detection of VKORC1 sequenceabnormalities, preferably based on the assay described in Example 9(ARMS PCR).

If the amount of expressed VKORC1 is to be determined, PCR primersspecific for a VKORC1 nucleic acid will be utilized for diagnostic ortherapeutic purposes. To this end RT-PCR technique, preferablyquantitative RT-PCR, may be carried out, wherein upon isolation of totalor mRNA from the sample the RNA is reverse transcribed into cDNA andsubsequently subjected to a PCR reaction using the specific primersaccording to the invention. This technique is well known to the skilledworker. This opens up a further possibility of obtaining the describedVKORC1 nucleic acids, for example by isolation from a suitable gene orcDNA library, for example from a liver disorder-specific or liverspecific gene bank, with the aid of a suitable primer. A preferred setof PCR primers and condition for isolating a VKORC1 nucleic acid areprovided in Example 5. Examples of preferred PCR primers according tothe invention directed against SEQ ID No. 2, are the primers accordingto SEQ ID No. 53-70 and preferred conditions for using these PCR primersare provided in FIG. 8.

The term “sample” is intended to refer to a biomaterial comprising fetalor adult tissue or cell, preferably tissue or cells, preferably isolatedor derived from heart, kidney and lung, pancreas, brain, placenta andskeletal muscle and blood, preferably from liver. The sample can beisolated from a patient or another subject by means of methods includinginvasive or non-invasive methods. Invasive methods are generally knownto the skilled artisan and comprise for example isolation of the sampleby means of puncturing, surgical removal of the sample from the openedbody or by means of endoscopic instruments. Minimally invasive andnon-invasive methods are also known to the person skilled in the art andinclude for example, collecting body fluids such as blood, preferably byvenopuncture, or urine or feces. The term “sample” may also encompass agenomic or an expression library, preferably constructed based on ansample isolated from a patient, in which case techniques for isolationof the cDNA that are generally known to the skilled worker may be used.

In another aspect the present invention provides a small interfering RNAmolecule (siRNA) and/or a short hairpin RNA (shRNA) directed against theVKORC1 nucleic acid, preferably against a sequence derived from SEQ IDNo. 1, 12, 17, 21, 25, and 27, or a nucleic acid according to SEQ ID No.2, 13, 18, 22, 26, and 28, which allows decreasing the stability of theVKORC1 nucleic acid and/or inhibiting the translation of the VKORC1nucleic acid in a cell culture or in vivo. The double-stranded siRNAsmediate sequence-specific, post-transcriptional silencing of a gene'sexpression by double-stranded RNA. The siRNAs have a very specificstructure: 17 to 25, preferably 19, 20, 21, 22, 23, 24, or 25nucleotides double stranded RNAs with 2 nucleotides 3′-end overhangs.siRNAs are usually derived from longer double stranded RNA molecules byenzymatic cleavage but siRNAs can also be synthesized chemically orenzymatically outside of cells and then delivered to cells (e.g., bytransfection). Thus, using siRNA or shRNA the expression of thecorresponding genes in cells can be decreased or even silenced both invivo and in vitro (McManus et al. 2002). shRNAs consist of a first stemportion comprising (I) a sequence of at least 18, preferably a least 19,more preferably at least 20 nucleotides that is complementary to themRNA sequence of a VKORC1 nucleic acid, preferably a sequencecomplementary to the SEQ ID No. 2, 13, 18, 22, 26, and 28; and (II) asecond stem portion comprising a sequence of at least 18, preferably aleast 19, more preferably at least 20 nucleotides that is sufficientlycomplementary to the first stem portion to hybridize with the first stemportion to form a duplex stem; and (III) a loop portion that connectsthe two stem portions. The loop portion may comprise at least 4,preferably at least 7, more preferably at least 11 nucleotides. ThesiRNA or shRNAs may also be included into a vector allowing constitutiveexpression of the siRNA or shRNAs in the host cell upon transformationof the host cell (cf. WO 03/006477). Strategies to the design of siRNAor shRNAs sequences and methods of constructing and producing thesemolecules are generally known to the person skilled in the art (cf.McManus et al., supra). The siRNA and shRNA molecules according to thepresent invention are for example useful for therapeutic regulation ofVKORC1 gene expression and for inclusion into methods for identifyingcoumarin derivatives, e.g. as a positive control for coumarin action.Preferred examples of siRNA sequences according to the invention arelisted in FIGS. 6 and 7 and are selected from SEQ ID No. 29, 20, 33, 34,37, 38, 41, 42, 45, 46, 47, 50. FIG. 6 also provides the respectivedownstram primers of these siRNA molecules which can be used forintegration into vectors so that the siRNA may be expressed in a celltargeted for siRNA expression.

As an alternative approach to silencing VKORC1 activity and/orexpression, the present invention provides antisense oligonucleotidesdirected against the VKORC1 nucleic acid as defined above, preferablyaccording to SEQ ID No 2, 13, 18, 22, 26, and 28, preferably against asequence derived from SEQ ID No. 1, 12, 17, 21, 25, and 27 (Zheng &Kemeny, 1995; Nellen & Lichtenstein, 1993).

According to another aspect of the present invention there is providedan RNA-aptamere directed against a VKORC1 polypeptide, preferablyagainst a sequence according to SEQ ID No. 1, which RNA-aptamere exertsan effect on the activity of the VKORC1 polypeptide. RNA-aptameres areeffective agonists or antagonists of proteins that are targeted by theaptameres, as has been shown for the coagulation factor IXa (Rusconi etal. 2002). An aptamere according to the invention may be used as ananticoagulant by reducing VKORC1-activity in a more fine-tuned mannerthan the coumarins. For testing aptameres they can be added to aVKORC1-reaction and analyzed by HPLC as described in Example 7.

In another aspect of the present invention there is provided an antibodywhich specifically recognizes and binds a VKORC1 polypeptide as definedabove, preferably a VKORC1 polypeptide according to SEQ ID No. 1, 12,17, 21, 25, and 27, or a fragment of the antibody. The antibody orantibody fragment preferably is a polyclonal or a monoclonal antibody,specific for the VKORC1 polypeptides. The antibody or antibody fragmentis produced according to methods generally known to the person skilledin the art by immunizing a mammal, for example a rabbit, with a VKORC1nucleic acid, or with a VKORC1 polypeptide according to the invention orparts thereof having at least 6 amino acid length, preferably having atleast 8 amino acid length, in particular having at least 12 amino acidlength, if appropriate in the presence of, for example, Freund'sadjuvant and/or aluminum hydroxide gels (see, for example, Harlow &Lane, 1998). The polyclonal antibodies formed in the animal as a resultof an immunological reaction can then be easily isolated from the bloodaccording to generally known methods and purified, for example, by meansof column chromatography. Monoclonal antibodies can be produced, forexample, according to the known method of Winter & Milstein (1991). Theantibodies according to the present invention can for example be usedfor diagnosis of VKORC1 associated deficiencies. Moreover, theantibodies may be useful for elucidating coumarin-VKORC1 interactions.Finally the antibodies may be used to isolate and/or purify VKORC1polypeptide from a tissue or cell sample isolated from a patient.

According to the present invention, the term “antibody” or “antibodyfragment” is understood as also meaning antibodies or antigen-bindingparts thereof prepared by genetic engineering and optionally modified,such as, for example, chimeric antibodies, humanized antibodies,multifunctional antibodies, bi- or oligospecific antibodies,single-stranded antibodies, F(ab) or F(ab)₂ fragments (see, for example,EP-B1-0 368 684, U.S. Pat. No. 4,816,567, U.S. Pat. No. 4,816,397, WO88/01649, WO 93/06213, WO 98/24884).

In another aspect the present invention provides a method of identifyinga coumarin derivative which exerts an effect onto the activity of VKORC1polypeptide as defined above, preferably the VKORC1 polypeptide havingthe sequence selected from SEQ ID No. 1, 12, 17, 21, 25, and 27,comprising the steps of:

-   (I) providing a host cell having been introduced the VKORC1 nucleic    acid or a vector containing the VKORC1 nucleic acid;-   (II) expressing the VKORC1 polypeptide in the host cell;-   (III) administering a candidate coumarin derivative;-   (IV) determining the activity of VKORC1 polypeptide (candidate    activity value);-   (V) comparing the candidate activity value with a control activity    value; and-   (VI) identifying the candidate coumarin derivative as a coumarin    derivative exerting an effect onto the activity of the VKORC1    polypeptide, provided the candidate activity value is significantly    different from the control activity value.

The determined activity of VKORC1 polypeptide isdithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitaminK quinone and wherein the significantly different activity value is acandidate activity value which is significantly higher than the controlactivity value, as described in further detail above and in Example 7and FIG. 10. If essentially the same concentration of candidate coumarinderivative yields a lower percentage of vitamin K epoxide converted intovitamin K quinone (product/substrate+product) as warfarine does in thisconcentration, this is indicative of the candidate coumarin derivativehaving a stronger inhibitory effect than warfarin, and vice versa.

Preferably in the method of identifying a coumarin derivative accordingto the present invention the control activity value is determined by amethod comprising the steps of:

-   (A) providing a host cell according to step (I);-   (B) expressing the VKORC1 polypeptide in the host cell; and-   (C) determining the activity of VKORC1 polypeptide (control activity    value).

Even more preferably in the method of identifying a coumarin derivativeaccording to the present invention at least one additional compound isintroduced into the host cell, which compound is selected from the groupconsisting of vitamin K, cytochrome B5, a nucleic acid coding forgamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, forcalumenin, or for glutathion-5-transferase. Methods for introducingnucleic acids into host cells have been described in detail above.Preferably the nucleic acids are expressed under the control of aconstitutively active promoter or a promoter which can be specificallyactivated in the host cell chosen.

The methods of identifying a coumarin derivative are useful fordeveloping novel coumarin derivatives that avoid at least one of thelimitations of coumarin and its derivatives known in the art. Ifanalysis of the kinetics of blood coagulation is included as a separateassay into the determination of VKORC1 polypeptide activity, the methodaccording to the invention may be useful in identifying coumarinderivatives which mediate blood coagulation faster than coumarin and itsderivatives known in the art and/or that are metabolized more rapidly sothat accumulation of coumarin and its derivatives may be prevented orameliorated and as a result the danger of overdosing is substantiallydecreased or even abolished. Moreover, such method of identifying may becombined with other assays such that coumarin derivatives may beidentified which have a stronger (weaker) effect onto VKORC1 activityand thus in turn onto the blood coagulation and which coumarinderivatives in independent assays prove (1) to be metabolized morerapidly so that accumulation of coumarin may be prevented or amelioratedand as a result the danger of overdosing is substantially decreased oreven abolished, (2) not to cause or to cause to lesser extend skinnecrosis in patients or embryopathy if applied during pregnancy, and/or(3) to be metabolized faster or to be less more stabil and/or to beaffected less by other drugs like Phenobarbital or amiodarone. Theassays which are suitable to screen for such properties of the coumarinderivatives and which are to be combined into a screen with the methodof identifying a coumarin derivative according to the invention aregenerally known to the person skilled in the art.

The term “coumarin” is understood as meaning3-(acetonylbenzyl)-4-hydroxycoumarin, i.e. COUMADIN® or sodium warfarin.

The term “derivative of coumarin” is understood to encompass organic orinorganic compounds, peptides, polypeptides or complexes thereof,provided that they exert an effect onto the activity and/or expressionof VKORC1 polypeptide, preferably an effect that inhibits the activityof the VKORC1 polypeptide, even more preferably a VKORC1polypeptide-specific effect, i.e the coumarin derivative does notdirectly interact with other molecules involved in the coagulationpathway. Examples of such compounds are organic molecules that arederived from libraries of compounds, preferably those that have beenanalyzed for their pharmacological activity. On account of theirinteraction, the derivatives of coumarin can influence the activity ofthe VKORC1 polypeptide in vivo and/or in vitro and enter intointeractions of covalent or non-covalent manner with them. If thecoumarin derivative is a chiral compound it is understood that“derivative of coumarin” also encompasses the respective R- andL-enantiomeril forms of the compound like those disclosed in WO00/43003. In particular the term “derivative of coumarin” refers tocompounds derived from 4-hydroxycoumarin, especially compounds derivedfrom COUMADIN. More preferably, “derivative of coumarin” also includesany coagulants which inhibits the regeneration of active vitamin K.

The term “candidate coumarin derivative” is understood to encompassorganic or inorganic compounds, peptides, polypeptides or complexes.Examples of such compounds are organic molecules that are derived fromlibraries of compounds, preferably those that have been analyzed fortheir pharmacological activity. Preferably the term refers to compoundsthat are structurally related or derived from 4-hydroxycoumarin,especially compounds related or derived from COUMADIN. If the candidatecoumarin derivative is a chiral compound it is understood that therespective R- and L-enantiomeric forms of the compound like thosedisclosed in WO 00/43003 are also encompassed by the term “candidatecoumarin derivative”.

In another aspect the present invention provides a method of determininga VKORC1 polypeptide sequence which conveys a coumarin effect exertedonto VKORC1 activity, comprising the steps of:

-   (I) providing a cell expressing the VKORC1 polypeptide according to    the invention, preferably a polypeptide according to SEQ ID NO. 1,    12, 17, 21, 25, and 27, which VKORC1 polypeptide has at least one    sequence abnormality, preferably a sequence abnormality selected    from the group consisting of V29L, V45A, R58G, R98W, L128R and    Y139C;-   (II) administering coumarin or a derivative thereof to the cell;-   (III) determining the activity of the VKORC1 polypeptide (sequence    abnormality activity value); and-   (IV) comparing the sequence abnormality activity value with the    control sequence activity value,    wherein a significant deviation of the sequence abnormality activity    value from the control sequence activity value is indicative that    the sequence abnormality of the VKORC1 polypeptide conveys the    coumarin effect exerted onto VKORC1 polypeptide. The activity of the    VKORC1 polypeptide may be determined as described in detail above.

More preferably, in the method of determining a VKORC1 polypeptidesequence the control sequence activity value is determined by a methodcomprising the steps of:

-   (I) providing a cell expressing the VKORC1 polypeptide, preferably a    polypeptide according to SEQ ID NO. 1, 12, 17, 21, 25, and 27;-   (II) administering coumarin or a derivative thereof to the cell;-   (III) determining the activity of the VKORC1 polypeptide (control    sequence activity value).

The determined VKORC1 activity is dithiothreitol-dependent conversion ofvitamin K 2,3-epoxide to vitamin K quinone and the significantlydifferent value is a sequence abnormality activity value which issignificantly higher than the control sequence activity value. Furtherdetails are provided above and in Example 7 and FIG. 10. The method maybe useful in identifying VKORC1 polypeptides that are less sensitive tocoumarins. By introducing VKORC1 polypeptides with different sequenceabnormalities this method allows identification of sites of thepolypeptide which are critical for the interaction of a tested coumarinand VKORC1. Such knowledge will for example be useful for designing newcoumarins.

It is particularly preferred in the method of determining a VKORC1polypeptide sequence that at least one additional compound is introducedinto the cell which compound is selected from the group consisting ofvitamin K, cytochrome B5, and a nucleic acid coding forgamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, forcalumenin, or for glutathion-5-transferase.

A further aspect of the present invention relates to a VKORC1polypeptide as defined above, preferably to the VKORC1 polypeptideaccording to SEQ ID No. 1, wherein the VKORC1 polypeptide contains atleast one sequence abnormality, which exerts an effect on the activityof the VKORC1 polypeptide. More preferably the VKORC1 polypeptide is thepolypeptide according to SEQ ID No. 1 and the sequence abnormality isselected from the group consisting of V29L, V45A, R58G, R98W, L128R andY139C. In another embodiment, the invention relates to the Rattusnorvegicus VKORC1 polypeptide according to SEQ ID No. 12 having asequence abnormality, preferably the sequence abnormality Y139C(416A>G). In another embodiment the invention relates to a Rattusnorvegicus nucleic acid encoding the Rattus norvegicus VKORC1polypeptide according to SEQ ID No. 12 having a sequence abnormality,preferably the VKORC1 nucleic acid according to SEQ ID No. 12 having asequence abnormality, preferably the 416A>G sequence abnormality. Thenucleic acid VKORC1 sequence containing the abnormality is 416A>G is thenucleic acid sequence according to SEQ ID No. 14.

Such VKORC1 polypeptide containing at least one sequence abnormality canbe generated by methods generally known to the skilled worker, includingrecombinant techniques and e.g. site directed mutagenesis, or byisolation the VKORC1 polypeptide having the at least one sequenceabnormality from a sample obtained from a patient, preferably from apatient suffering from VKORC1 associated deficiencies. Methods forisolating proteins from a sample have been described in detail above.

The VKORC1 polypeptide containing at least one sequence abnormality,which exerts an effect on the activity of the VKORC1 polypeptide can,for example, be used for generating antibodies binding specifically tothese VKORC1 polypeptides. These antibodies in turn can be utilized fordiagnosing VKORC1 associated deficiencies.

The term “sequence abnormality” is meant to encompass additions,insertions, deletions, substitutions of at least one amino acid thatresult in an alteration of the VKORC1 polypeptide sequence, preferablyof the VKORC1 polypeptide sequence according to SE ID No. 1. Alsoencompassed are additions, insertions, deletions, substitutions of atleast one nucleotide that lead to an altered amino acid sequence encodedby the VKORC1 nucleic acid sequence. Also encompassed are changes of theVKORC1 nucleic acid sequence that lead to a change in the reading frameof the nucleic acid sequence.

The sequence abnormalities are indicated in the single letter amino acidcode with the original amino acid being placed to the left of the numberindicating the number of the amino acid in the polypeptide sequenceaccording to SEQ ID No. 1. The number to the right of the amino acidnumber indicates the amino acid that replaces the original amino acid.For example, the sequence abnormality V29L indicates that in position 29of the VKORC1 polypeptide of SEQ ID No. 1 the amino acid valine (V) hasbeen replaced by leucine (L). In case the sequence abnormality occurs ina VKORC1 polypeptide other than the VKORC1 polypeptide of SEQ ID No. 1the number in the code refers to the sequence position according to thenumbering of the amino acids in that particular other polypeptide.

In another aspect the present invention provides a VKORC1 nucleic acidselected from the group consisting of:

-   (a) a nucleic acid coding for the VKORC1 polypeptide containing at    least one sequence abnormality, which exerts an effect on the    activity of the VKORC1 polypeptide, wherein the VKORC1 polypeptide    is preferably the polypeptide according to SEQ ID No. 1 and the    sequence abnormality is selected from the group consisting of V29L,    V45A, R58G, R98W, L128R and Y139C;-   (b) a nucleic acid sequence selected from the group consisting of a    sequence according to SEQ ID No. 3, 4, 5, 6, and 7, 14, and 94;    and (c) a nucleic acid sequence which, but for the degeneracy of the    genetic code, would hybridize to the nucleic acid defined in (a)    or (b) and which nucleic acid sequence codes for the polypeptide    containing at least one sequence abnormality as defined above.

These nucleic acids are for example useful for isolating VKORC1polypeptides and nucleic acids having such sequence abnormality, forproducing DNA and/or RNA probes, for producing antibodies, for theconstruction of transgenic animals and knock-out animals, and forinclusion into screens for identifying coumarin derivatives.

In a further aspect the present invention provides a vector containingthe VKORC1 nucleic acid containing at least one sequence abnormality asdefined above. Such vectors may be selected from the vectors describedin detail above. The methods for constructing such vectors are alsodescribed in detail above. Such vectors are for example useful forpreparing probes described in the next paragraph, especially in adiagnostic context.

In another aspect of the present invention there is provided a DNA or aRNA probe directed against VKORC1 nucleic acid containing at least onesequence abnormality as defined above, preferably a nucleic acidsequence according to SEQ ID No. 3, 4, 5, 6, and 7, 14, and 94. Methodsof designing and producing such DNA and RNA probes have been describedto a great extend above. Such probes are useful for example fordetecting sequence abnormalities in the VKORC1 gene in a gene library,in an expression library, or in a sample isolated from a patient, forexample in the context of diagnosis of VKORC1 associated deficienciesand for site directed mutagenesis for producing VKORC1 nucleic acidscontaining a sequence abnormality. Techniques for screening librariesare generally known to the skilled worker.

In another aspect of the present invention there is provided a PCRprimer directed against VKORC1 nucleic acid containing at least onesequence abnormality as defined above. Preferred PCR primers fordetecting the Y139C (416A>G) sequence abnormality are the primersaccording to SEQ ID No. 88 to 91 (see Example 9). It is generally knownto the person skilled in the art to design the PCR primers such thatthey may be used for detecting other sequence abnormalities such asthose mentioned above (V29L, V45A, R58G, R98W, L128R) for the purpose ofthe invention. Methods of designing and producing such PCR primers,techniques to carry out PCR amplification have been described to a greatextend above, the major difference being that the primers have to bedesigned such that only those VKORC1 nucleic acid sequences containingthe sequence abnormality are specifically amplified, whereas nativeVKORC1 nucleic acids and other VKORC1 nucleic acid sequences that do notcontain the sequence abnormality, such as the VKORC1 nucleic acid of SEQID No. 2 remains undetected. Such primers are useful for example fordetecting sequence abnormalities in the VKORC1 gene in a gene library,in an expression library, or in a sample isolated from a patient, forexample in the context of diagnosis of VKORC1 associated deficiencies.Techniques for screening libraries are generally known to the skilledworker.

In a further aspect the present invention relates to an antibody whichspecifically recognizes and binds the VKORC1 polypeptide containing atleast one sequence abnormality as defined above, the VKORC1 polypeptidepreferably being the polypeptide according to SEQ ID No. 1 and thesequence abnormality being selected from the group consisting of V29L,V45A, R58G, R98W, L128R, and Y139C, or a fragment of the antibody. Thetypes of antibodies encompassed, methods of constructing and producingsuch antibodies and fragments thereof have been described in greatdetail above. Such antibodies are e.g. useful for the detection andisolation of VKORC1 polypeptide containing at least one sequenceabnormality as defined above, especially in the context of diagnosis ofVKORC1 associated deficiencies and for detection of Warfarine resistancein humans and rodents such as rats.

According to another aspect the present invention relates to adiagnostic comprising a compound selected from the group consisting ofthe VKORC1 nucleic acid containing at least one sequence abnormality,preferably at least one sequence abnormality selected from V29L, V45A,R58G, R98W, L128R, and Y139C; the DNA or the RNA probe directed againstthe VKORC1 nucleic acid containing at least one sequence abnormality,the PCR primer directed against the VKORC1 nucleic acid containing atleast one sequence abnormality, and an antibody directed against theVKORC1 polypeptide containing at least one sequence abnormality; all ofwhich have been defined above. In the case the VKORC1 associateddeficiency is due to or correlated with a sequence abnormality, it isthe principle of the diagnostic to be used for detection of thatsequence abnormality in a probe obtained from a patient. The suitablemethods for using the diagnostic according to the invention arementioned below. Optionally the diagnostic further comprises apharmaceutically acceptable additive and/or auxiliary. Such diagnosticis useful for diagnosing VKORC1 associated deficiencies especiallywarfarin resistance.

In on the other hand the VKORC1 associated deficiency is to be diagnosedbased on the detection of the level of expression of VKORC1 mRNA, VKORC1cDNA or VKORC1 polypeptide in the sample, such levels of expression canbe determined by methods generally known to the person skilled in theart. Examples of such methods for detecting the presence of a VKORC1mRNA include RNA blot (Northern) analysis, nuclease protection, in situhybridization, reverse transcriptase PCR (RT-PCR; including quantitativekinetic RT-PCR). cDNA and oligonucleotide microarrays are also includedas such methods. An expression library derived from a patient may aswell be screened for the purpose of diagnosis using techniques generallyknown to the skilled worker. The presence of VKORC1 polypeptide can alsobe determined by methods generally known to the skilled worker, some ofwhich are described below. Optionally the diagnostic further comprises apharmaceutically acceptable additive and/or auxiliary.

Within the meaning of the present invention “additive” and “auxiliary”are not particularly limited and generally known to the person skilledin the art and comprise, for example, physiological saline solution,demineralized water, gelatin or glycerol-based protein stabilizingreagents. Alternatively, the VKORC1 nucleic acids, probes, primers orpolypeptide according to the present invention may be lyophilized forstabilization.

In another aspect the invention provides a method of diagnosing a VKORC1associated deficiency in a patient comprising the steps of:

-   (I) amplifying a DNA sample obtained from the patient or reverse    transcribing a RNA sample obtained from the patient into a DNA and    amplifying the DNA; and-   (II) analyzing the amplified DNA of step (I) to determine at least    one sequence abnormality in a nucleic acid sequence coding for the    VKORC1 polypeptide or in an amino acid sequence of the VKORC1    polypeptide;    wherein the determined sequence abnormality is indicative of the    patient suffering from a VKORC1 associated deficiency; preferably    Warfarine resistance; preferably the sequence abnormality exerts an    effect on the activity of the VKORC1 polypeptide, preferably the    sequence abnormality is selected from V29L, V45A, R58G, R98W, L128R,    and Y139C.

Methods for obtaining samples from a patient and for isolating total RNAor mRNA are generally known to the skilled worker, some of which havebeen described above. Techniques for amplifying of DNA are notparticularly limited and include PCR techniques which have also beendescribed above. By the same token, techniques for reverse transcribinghave been mentioned above and are not particularly limited and includereverse transcription using conventional protocols and commerciallyavailable kits that usually employ reverse transcriptase and oligo dTprimers. The analysis may as well be based on genomic DNA isolated froma sample obtained from a patient.

Upon amplification, the DNA is subjected to analysis in order todetermine at least one sequence abnormality in a nucleic acid sequencecoding for the VKORC1 polypeptide. Methods for analyzing the amplifiedDNA are not particularly limited. Preferably the amplified DNA isanalyzed by a technique selected from the group consisting of PCR-basedanalysis, preferentially using PCR primers specific for the sequenceabnormality, restriction digestion analysis, and DNA sequencinganalysis. In a preferred embodiment the nucleic acid carrying thesequence abnormality is coding for a VKORC1 sequence having a sequenceabnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), Y139C(416 A>G). In a preferred embodiment of the method of diagnosing theamplified DNA encodes at least a partial sequence of the is VKORC1polypeptide according to SEQ ID No. 1. One way of determining a sequenceabnormality which is associated with a VKORC1 associated deficiency isprovided in Example 8.

The VKORC1 sequences according to the invention containing the mutations85 G>T, 134 T>C, 172 A>G, 292 C>T, and 383 T>G, are provided in SEQ IDNos. 3 to 7 and may be used as probes for diagnosing a VKORC1 associateddeficiency using hybridization technique based analysis of nucleic acidsamples obtained from a patient.

As an alternative VKORC1 expression may be detected on the level of theVKORC1 polypeptide. Therefore, in another aspect the present inventionprovides a method of diagnosing a VKORC1 associated deficiency in apatient comprising the steps of:

-   (I) providing a sample obtained from the patient; and-   (II) detecting a VKORC1 polypeptide having a sequence abnormality in    the sample using the antibody directed against the VKORC1    polypeptide having a sequence abnormality as defined above,    wherein the determined sequence abnormality is indicative of the    patient suffering from a VKORC1 associated deficiency. Preferably    the sequence abnormality is selected from the group consisting of    V29L, V45A, R58G, R98W, L128R, and Y139C.

Methods for obtaining samples have been described in detail already.Methods for detection of VKORC1 polypeptide having a sequenceabnormality are not particularly limited, provided that the methodallows specific detection of the protein carrying the sequenceabnormality(ies). Examples of such methods preferably includeimmunohistochemical detection, immunoblotting, preferably Westernblotting, and ELISA of the polypeptide, particularly preferred withantibodies specific for the VKORC1 polypeptide having a sequenceabnormality as defined above. Analysis of sequence abnormalities on thelevel of the amino acid sequence is not particularly limited and may forexample be carried out using VKROP1 antibodies which specificallyrecognize and bind a VKORC1 polypeptide having one or more sequenceabnormalities.

Preferably the methods of diagnosing are used for diagnosing diseasesand/or disorder selected from warfarin resistance, familial multiplefactor deficiency, a disorder or disease associated with reduced orabolished blood coagulation, such as hemophilia and a disorderassociated with decreased vascular calcification, diseases and/ordisorders associated with impaired gamma-carboxylation of vitamin Kdependant proteins.

Moreover, the methods of diagnosing may also be used to diagnosedisorders and diseases associated with increased blood coagulationincluding patients and/or patients having an increased risk ofdeveloping a thrombus due to a sequence abnormality in the VKORC1polypeptide or its gene, diseases and/or disorders associated withimproved gamma-carboxylation of vitamin K dependant proteins.

In another aspect the present invention provides a method of identifyinga coumarin derivative which exerts an inhibitory effect onto theactivity of a VKORC1 polypeptide having at least one sequenceabnormality, comprising the steps of:

-   (I) providing a cell expressing the VKORC1 polypeptide, preferably a    polypeptide encoded by a sequence selected from the group consisting    of 3, 4, 5, 6, 7, 14, and 94;-   (II) administering a candidate coumarin derivative to the cell;-   (III) determining the activity of the VKORC1 polypeptide (sequence    abnormality activity value); and-   (IV) comparing the sequence abnormality activity value with the    control sequence activity value,-   (V) identifying the candidate coumarin derivative as the coumarin    derivative exerting an inhibitory effect onto the activity of a    VKORC1 polypeptide, if the administration of the candidate coumarin    derivative results in a sequence abnormality activity value which is    significantly lower than the control sequence activity value.

More preferably, the control sequence activity value is determined by amethod comprising the steps of:

-   (I) providing a cell expressing the VKORC1 polypeptide of claim 1,    preferably a polypeptide according to SEQ ID No. 1 or 12;-   (II) administering coumarin to the cell;-   (III) determining the activity of the VKORC1 polypeptide (control    sequence activity value).

The activity of the VKORC1 polypeptide may be determined as described indetail above. And the method may be adopted from the method described inExample 7. Such method of identifying a coumarin derivative is usefulfor identifying coumarin derivatives that can be used as anticoagulantsin warfarin resistance patients.

In another aspect the present invention provides a method of identifyinga coumarin derivative which is toxicologically effective inwarfarin-resistant rodents comprising the steps of:

-   (I) providing a warfarin-resistant rodent;-   (II) administering a candidate coumarin derivative to the rodent;-   (III) determining the toxicity of the candidate coumarin derivative    onto the rodent (candidate coumarin derivative toxicity value);-   (IV) comparing the candidate coumarin derivative toxicity value with    a control coumarin toxicity value; and-   (V) identifying the candidate coumarin derivative as a    toxicologically effective coumarin derivative, provided that the    candidate coumarin derivative toxicity value is significantly larger    than the control coumarin toxicity value.

Preferably, the warfarin-resistant rodent is a rodent transgenic forVKORC1 polypeptide as defined above, wherein the VKORC1 polypeptidecontains at least one sequence abnormality, which causes warfarinresistance, preferably a polypeptide encoded by the sequence accordingto SEQ ID No. 14 or a commercially is available Warfarine resistant rator a wild catch rat having Warfarine resistance. More preferably, theVKORC1 polypeptide is the polypeptide according to SEQ ID No. 12 and thesequence abnormality is selected from the group consisting of V29L (85G>T), V45A (134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383T>G), and Y139C (416 A>G).

The sample can be any organ, tissue, body fluid or probe provided thatit contains genomic DNA or mRNA from the rat to be tested. Preferablythe sample is blood, tissue from tail or ear urine or feces. Furtherdetails of the method are provided in Example 9.

Warfarin-resistant rodents have been described (Kohn & Pelz, 1999) andmay be obtained from commercial suppliers (e.g. The Federal BiologicalResearch Center for Agriculture and Forestry, Institute for Nematologyand Vertebrate Research, Toppheideweg 88, 48161 Münster, Germany). In apreferred embodiment the warfarin-resistant rodent is a rodenttransgenic for VKORC1 polypeptide containing at least one sequenceabnormality, which sequence abnormality causes warfarin resistance. Morepreferred, the VKORC1 polypeptide containing at least one sequenceabnormality is the polypeptide encoded by the nucleic acid sequenceaccording to SEQ ID Nos. 3 to 7, and 15, and the sequence abnormality isselected from the group consisting of V29L (85 G>T), V45A (134 T>C),R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), Y139C (416 A>G).Methods for generating rodent, in particular mice transgenic for therecited VKORC1 polypeptides have been described above.

According to another aspect the invention relates to the use of PCRprimers according to SEQ ID No. 88 to 91 for determining whether or nota rat has a warfarin resistance genotype in a sample obtained from arat.

The administration of coumarin and its derivatives is not particularlylimited. Typically coumarin (warfarin)-containing toxicologicallyeffective compositions are formulated as granular bait compositionscontaining from about 50-300 ppm, preferably about 250 ppm, of coumarinand its derivatives. The bait is typically formulated with from 0.5% to2.5% of warfarin concentrate in a suitable binder such as corn oil. Ifcorn oil is used as a binder, it can be present in an amount of fromabout 0.5% to about 2% of the total composition. The binder and warfarinare then mixed in e.g. with a product based on cereal products, cornmeal, rolled oats, mixed animal feeds, and similar products known in theart. The administration, i.e. the amount, formulation and frequency andduration of administration may follow standard protocols for assessingthe toxicity of warfarin in rodents, more preferred follow standardprotocols for assessing the lethal dose 50 (LD₅₀) value for a givenpoison to be tested, all of which are generally known to the skilledperson and described in example 9

In order for the coumarin derivative to be toxicologically effective itis desirable that multiple ingestions are required to kill the rodent sothat they do not develop bait shyness. Therefore, it is preferred torepeat the administration of the candidate coumarin derivativecompositions a number of times. Usually the rodents begin to die afterfour or five daily doses of the compositions. Moreover, it may bepreferred to suppress pain in the rats in order to ameliorate thesuffering during the experiments by administration of pain suppressingagents generally known to the skilled worker. Inclusion ofpain-suppressors in the coumarin and coumarin derivative composition mayfurther be advantageous in order to further suppress the chance thatrodents develop bait shyness.

Following administration the toxicity of the candidate coumarinderivative onto the rodent is determined which yields the candidatecoumarin derivative toxicity value. Methods for determining the toxicityof candidate coumarin derivatives are generally known to the skilledworker and include LD₅₀ analysis, analysis of the blood coagulation bydetermining the prothrombin time, e.g. by international normalizedration (INR) protocol. The determined candidate coumarin derivativetoxicity value is then compared with an appropriate control coumarintoxicity value determined on the basis of subjecting a differentspecimen of the warfarin-resistant rodent to the same treatment butexchanging the coumarin derivative administration with an administrationof a standard rodent coumarin composition which the rodents areresistant for, commonly used for pest control. The same experimentalconditions described above for the candidate coumarin derivativeadministration are used for the control. If the candidate coumarinderivative toxicity value is equal or preferably statisticalsignificantly larger than the control coumarin toxicity value, thecandidate coumarin derivative represents a toxicologically effectivecoumarin derivative.

In another aspect of the invention the invention provides a compositionfor killing rodents, comprising a toxicologically effective amount ofthe coumarin derivatives identified by the method described above. Theformulation of bait compositions containing coumarin and its coumarinderivative have been described above. A typical formulation has thefollowing constituents: Ingredient %: Grain carrier 94%, Corn oil 1.0%,coumarin or coumarin derivative concentrate (0.5%) 5.0; total 100.0%

The invention further relates to the following embodiments: A coagulantpharmaceutical composition comprising a compound selected from the groupconsisting of the VKORC1 polypeptide, the VKORC1 nucleic acid, thefusion protein according to the invention, the vector according to theinvention, the host cell according to the invention, optionally combinedwith a pharmaceutically acceptable carrier.

A method of treating a patient in need of such treatment comprising thestep of administering to the patient a therapeutically effective amountof the coagulant pharmaceutical composition. The method can be used fortreating a patient suffering from a VKORC1 associated deficiency.

An anticoagulant pharmaceutical composition comprising a compoundselected from the group consisting of the siRNA and/or shRNA accordingto the invention, the antisense RNA or DNA according to the invention,the RNA-aptamere according to the invention, the antibody according tothe invention, optionally combined with a pharmaceutically acceptablecarrier

A method of treating a patient in need or such treatment comprising thestep of administering to the patient a therapeutically effective amountof the anticoagulant pharmaceutical composition.

The use of a VKORC1 polypeptide or a VKORC1 nucleic acid forgamma-carboxylating vitamin-K dependant polypeptides. Preferably thegamma-carboxylated vitamin-K dependant polypeptide is a polypeptideselected from the group consisting of blood coagulation factor II, VII,IX, X, protein C, protein S, protein Z, matrix gla protein, andosteocalcin. In a preferred embodiment the VKORC1 is used in combinationwith at least one additional compound preferably in a cellular setting,which additional compound is selected from the group consisting ofvitamin K, cytochrome B5, and a nucleic acid coding forgamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, forcalumenin, or for glutathion-5-transferase

The invention will now be further illustrated below with the aid of thefigures and examples, representing preferred embodiments and features ofthe invention without the invention being restricted thereto.

EXAMPLES Example 1 Characterization of the Genomic Candidate Region

The locus for combined deficiency of vitamin K-dependent clotting factortype 2 (VKCFD2) to the pericentromeric region of chromosome 16 betweenthe markers D16S3131 and D16S419 has been mapped [Fregin et al., 2002].This region comprises approximately 20 Mb. The genes responsible forwarfarin resistance in rats (Rw) and mice (War) had been mapped tochromosome 1 [Kohn et al., 1999] and chromosome 7 [Wallace,1976][Greayses & Ayres, 1967] in close linkage to the myosin light chain2 gene (Myl2). The human ortholog of Myl2, HUMMLC2B, is located onchromosome 16 μl within the VKCFD2 candidate region and is part of aconserved linkage group of genes. Based on this synteny and onbiochemical considerations, it is hypothesized that VKCFD2 and warfarinresistance may be due to allelic mutations in the same gene. If so, thiswould narrow down the critical interval in humans to a region ofapproximately 4.5 Mb between the interleukin 4 receptor gene (IL4R) andthe integrin alpha M chain gene (ITGAM) on the short arm of chromosome16 (FIG. 1).

According to the genome assembly, this region contains 141 Ensembl geneswith approximately 1000 exons. Of these genes, 117 were annotated asknown. Many of these genes could be excluded from further analysisbecause their function was well established and obviously not related tothe metabolic steps of the vitamin K cycle. On the other side, genesupstream and downstream of this region are included that were regardedas functional candidates into the mutation screen.

Example 2 Mutation Screening

Using genomic DNA from two VKCFD2 and three WR subjects, a systematicmutation screen was initiated by comparative sequencing of the remainingcandidate genes. Clinical data of the VKCFD2 families have beendescribed previously [Oldenburg et al., 2000]. Warfarin resistantpatients were ascertained due to their abnormal response to oralwarfarin administration during thrombosis treatment or prevention.Patient C and E are sporadic cases. Patient D has two brothers alsosuffering from warfarin resistance. Patients C and D requiredapproximately 150-250 mg warfarin per week to achieve a therapeuticrange of oral anticoagulation whereas patient E did not show anyresponse at all. All patients gave informed consent beforeparticipating.

Surprisingly, missense mutations were found in a gene of unknownfunction in all investigated VKCFD2 and WR subjects (FIG. 2). This gene(IMAGE3455200) spans a genomic region of 5126 bp and comprises threeexons coding for a protein of 163 amino acids. It was named vitamin Kepoxide recycling protein 1 (VKORC1). Both non-related VKCFD2 patientsand their affected siblings were found to harbor the same homozygouspoint mutation in the third exon (292C>T) whereas the parents were foundto be heterozygous. The mutation is caused by the replacement ofarginine by tryptophan at amino acid residue 98 (R98W). The families areof German and Lebanese origin. The haplotypes in the region ofhomozygosity around the mutated gene were different in both familiesindicating independent mutation events. In the WR patients, threedifferent heterozygous mutations were found leading to a valine byleucine substitution (patient C: V29L), an arginine by glycinesubstitution (patient D: R58G) and an exchange of leucine to arginine(patient E: L128R). The R58G mutation is shared by the two affectedbrothers of index patient D. The missense mutations were not present in384 control chromosomes. Sequencing of the control chromosomes revealedtwo non-synonymous single nucleotide polymorphisms (C43C; L120L).

Genome sequences and annotation were obtained from NCBI, UCSC andEnsembl. Primers for mutation screening were designed using Primer3software integrated into a script, ExonPrimer, to allow automatic primerdesign (http://ihg.gsf.de/ihg/ExonPrimer.html). For mutation screening,exons with intronic primers were amplified and amplified fragments wereanalyzed by direct sequencing with the BigDye Terminator Cyclesequencing kit (ABI)). Primer sequences were available on request.Topology predictions were performed using TMPRED and TMHM.

Example 3 Homology and Protein Structure

An orthologue of the VKORC1 gene was present in mouse (NM_(—)178600) andthe orthologues in rat and in Fugu rubripes were established by homologysearches and RT-PCR (FIG. 3). The corresponding proteins share 79% to84% identity with the human protein. Database searches did not show anyhomology to a known gene nor to any characterized protein domain.Topology prediction programs anticipated three transmembrane domains(TM). The first TM is placed between residues 10 to 29 by all programstested. The predictions are discordant for the second and the third TM,which are located between amino acids 100 and 150. The PSORT II serverpredicted an ER membrane retention signal (KKXX or KXKXX) at position159-163 of human VKORC1 with a probability of 67% [Jackson et al.,1990]. The consensus sequence was also present in the other VKORC1proteins. This is in accordance with the likely location of the VKORC1complex within the ER membrane system [Cain et al., 1997].

Tblastn searches with VKORC1 detected a homologous human (BC027734) andmouse gene (AK009497) showing 50% protein identity each. Both mRNA werewrongly predicted to code for proteins that show no homology withVKORC1. The predicted human protein starts at the third methionine. Themouse mRNA sequence is incomplete with a protein predicted in adifferent reading frame. The complete cDNA was established in mouse aswell as in Fugu rubripes and partially in rat. These proteins weredesignated VKORC1 like protein 1 (VKORC1L1). Human, mouse and ratVKORC1L1 proteins share approximately 84% identity between each otherand approximately 50% identity with the corresponding VKORC1 proteins. Ahomologous protein was further detected in Xenopus laevis (AAH43742)and—with weaker homology (1e-14)—in Anopheles gambiae (EAA06271). Treeanalysis suggested that both these proteins are orthologues to theVKORC1 gene.

Example 4 Expression Analysis

VKORC1 seems to be widely expressed. The corresponding Unigene entrycontains more than 100 ESTs in various tissues. The expression of VKORC1in fetal and adult human tissues is examined by Northern blot analysis.To this end Human multiple tissue northern blots (Fetal Blot 1,Stratagene; Human 12-Lane, BD Clontech) contained 2 μg of poly(A)+-RNA.Full-length human VKORC1 cDNA was radiolabeled using random primers DNAlabeling system (Invitrogen life technologies). and hybridized usingmiracleHyb High-Performance Hybridization Solution (Stratagene). A1′-actin probe supplied with the multiple tissue northern blot was usedfor control hybridization.

The highest VKORP expression levels can be observed in fetal and adultliver is (FIG. 4). High expression levels were also observed in fetalheart, kidney and lung, as well as in adult heart and pancreas. Fetalbrain, adult placenta and skeletal muscle showed intermediate levels ofexpression. Minor expression levels were detected in adult brain, lungand kidney.

Example 5 Cloning of VKORC1 and Construction of Expression Vectors

Amplification of the complete coding sequence of VKORC1 was performedfrom human liver and kidney cDNA (Marathon-Ready cDNA, BD BiosciencesClontech) with the following primers including cleavage sites forHindIII and EcoRI:

VKORC1-HindIII-F: (SEQ ID No. 53) ATTAAGCTTCACCATGGGCAGCACCTGGGGGAGCCCTVKORC1-EcoRI-R: (SEQ ID No. 54) ATTGAATTCCGTGCCTCTTAGCCTTGCCCTG.

The product was cloned into the pBluescript II vector (Stratagene) thatwas cleaved with the corresponding restriction enzymes and verified bydirect sequencing. For immunocytochemistry experiments, the insert wasre-cloned into the mammalian expression vectors pEGFP-N1 (BD BiosciencesClontech) and pcDNA3.1/myc-His (Invitrogen).

For expression studies, the VKORC1 cDNA was cloned into the pcDNA3vector (Invitrogen) after amplification with the primersVKORC1-pcdna3-F: GGGCGGAAGCTTGAGATAATGGGCA (SEQ ID No. 92) andVKORC1-pcdna3-R: GCTTGAATTCAGGGCTCAGTGC (SEQ ID No. 93). Mutagenesis wasperformed using the QuikChange mutagenesis Kit (Stratagene). Wildtypeand mutated cDNAs were re-cloned for expression in pCEP4 (Invitrogen)using the HindIII and XhoI-sites. All constructs were verified bysequencing.

Example 6 Cell Culture, Transient Transfection and Immunocytochemistryand Subcellular Localization

From biochemical fractionation experiments it is known that the VKORC1activity purifies with the microsomal membrane fraction [Cain et al.,1997]. Furthermore, the gamma-glutamyl-carboxylase has been localized tothe membrane of the endoplasmic reticulum by immunocytochemistry[Presnell, 2002 #31]. In order to study the subcellular localization ofhuman VKORC1 GFP- and myc-epitope tagged VKORC1 fusion proteinconstructs were generated for transient transfection experiments ofCOS-7 cells. Primary antibodies against the epitope tags andfluorochrome labeled secondary antibodies were used to visualize thefusion proteins. An antibody against the ER-specific protein calnexinserved as a control. To this end COS-7 cells (DSMZ, Braunschweig) weremaintained in Dulbecco's modified eagle's medium with 10% fetal calfserum. Cells were plated on glass cover slips in six-well plates andafter 18-24 h in culture transfected with the expression vectorconstructs using Effectene (QIAGEN) according to the manufacturer'sspecifications. After 48-60 h of further culturing, the cells werewashed with PBS and fixed in 70% acetone 130% methanol at −20° C. for 15min. Following fixation, the cells were permeabilized in PBS, 0.1%Nonidet P-40 (SIGMA N-6507), and then blocked with PBS, 2% BSA and 0.1%NP-40 at 37° C. Primary antibodies, Living Colors A.v. (JI-8) (BDBiosciences Clontech), anti-myc antibody (Invitrogen), and anti-calnexin(SIGMA) were diluted (1:100) in the blocking solution and incubated for45 min at 37° C. Cover slips were washed in PBS, 0.1% NP-40 for 30 min.The same incubation and washing procedures were used for the secondaryantibodies, i.e. anti-mouse-IgG-FITC (SIGMA) and anti-rabbit-IgG F(ab′)2fragment-Cy3 (SIGMA). Cover slips were counterstained with DAPI (1:500)for 1 min, washed with deionized water, mounted on slides usingVectashield (Vector) and visualized using a Leica fluorescencemicroscope.

The green immunofluorescence of the VKORC1 fusion proteins decorated themesh-like structures of the ER within the cytoplasm and perfectlyco-localized with the label of the ER-marker calnexin (red) (FIG. 5).

Example 7 An assay for Determining Enzymatic VKORC1 Activity

HEK293-EBNA cells (Invitrogen) were grown in MEM with 10% FCS. For eachexperiment, 6×10⁵ cells were plated onto 94 mm Petri dishes. After 30 hat 37° C. and 5% CO₂, transfection (20 μg of DNA construct per dish) wasperformed using the calcium phosphate method. After 40 h at 35° C., 3%CO₂ transfected cells (nearly grown to confluence) were washed in PBS,harvested and lysed in 450 μl 0.25 mM imidazole, (pH=7.6), 0.5% CHAPS.Transfection efficiency was checked by sequencing of RT-PCR products ofan aliquot of cells.

VKOR enzymatic activity was measured with 30 μl of the whole-cellextracts which were resuspended in 500 μl buffer A (0.25 mM imidazole,(pH=7.6), 0.5% CHAPS). Then 20 μl 125 mM DTT were added with one minuteincubation. Then 5 μl 400 mM CaCl₂, and Warfarin in 10 μl DMSO (finalconcentration 0-80 μM) were added. The reaction was started by additionof 2 μl vitamin K2,3-epoxide (final concentration 5 μM) and incubated at30° C. for one hour. Reaction was stopped by extraction of the substrate(Vitamin K2,3-epoxide) and the reaction products (Vitamin K-quinone andhydroquinone) using 1 ml 2-Propanol/Hexane (3:2, v/v); the organicsupernatant was collected, dried and resolved in 50 μl methanol andanalyzed with an HPLC at 254 nm. The vitamin K quinone was separatedfrom the epoxide by HPLC on a reversed phase C-18 column. During theextraction procedure vitamin K hydroquinone was quantitatively oxidizedto the quinone form. The output of the HPLC was analyzed automaticallyby calculating the area under the line of extinction of each peak. Thepercentage of conversion of substrate was estimated by setting the areaof the residual substrate-peak (epoxide) plus the product-peak (quinone)as 100 percent. Measurements were run in duplicate and the activity isgiven as percent of substrate converted into quinone. Vitamin K2,3-epoxide was prepared by oxidation of vitamin K quinone(Sigma-Aldrich) with H₂O₂. Warfarin (Sigma-Aldrich) was added in DMSO(<1 Vol %).

Dose-response to warfarin inhibition was measured at 5 to 80 μM finalconcentration (Walin & Martin 1985). Untransfected and mock-transfectedcells showed a low basal activity which was warfarin sensitive.Overexpression of Wildtype VKORC1 resulted in a striking stimulation ofVKOR activity. Production of vitamin K quinone was 14 to 21-foldincreased compared to untreated and mock-transfected cells. The activitywas inhibited by warfarin in a dose-dependent manner (FIG. 10).

We also determined VKOR activity after transfection with mutated VKORC1constructs (FIG. 10). Recombinant expression of the R98W mutationobserved in the two VKCFD2 families did only slightly increase VKORactivity in HEK293 cells. Spontaneous bleeding episodes and high serumlevels of vitamin K epoxide in these patients suggest that theefficiency of vitamin K recycling is also drastically decreased in vivo(Oldenburg et al. 2000). The five WR mutations showed a reduced VKORactivity ranging from 5% in the L128R variant to 96% in the V29Lmutation. Mutations V45A, R58G and Y139C displayed about 23%, 21% and48% activity, respectively (Table 1). Reduced VKOR activity associatedwith higher vitamin K demand and death from spontaneous bleeding hasbeen observed in heterozygous and homozygous Rw rats (Martin et al.1979, Thijssen & Pelz 2001, Fasco et al. 1983b). Similarly, in ourexpression system which mimics homozygous conditions WR mutations led toa lower functional efficiency of the VKOR complex. Whereas at thephenotypic level all WR variants exhibited at least partial resistancetowards the anticoagulation effect of warfarin, both Wildtype and mutantproteins were sensitive to warfarin in vitro. At concentrations above 20μM, mutations V29L and Y139C retained higher VKOR activities than theWildtype while in mutations V45A, R58G, L128R, VKOR activity fell belowthe detection limit (FIG. 10).

Example 8 A method of Diagnosing a VKORC1 Sequence Abnormality

Genomic DNA of the specimen (human patient or mammal) is isolatedaccording to standard procedures generally known to the skilled worker.The genomic DNA of the desired Exon (1-3) of VKORC1 is amplified by PCRusing specific primers which can also be designed by the skilledartisan. The PCR-product is then purified using e.g. SAP/Exo (shrimpalkaline phosphatase and exonuclease) under standard conditions.

The purified DNA is then subjected to standard sequencing proceduressuch as: addition of 0.3 μl primer which is 10 pmol/μl (forward orreverse primer) to 1 μl of the purified PCR-product; followed byaddition of 8 μl DTCS-Mix (Beckman-Coulter) and 10.7 μl water; followedby cycle sequencing at

First delay 96° C. 60 sec Denaturation 95° C. 30 sec 30 Cycles AnnealingPrimerspecific(55-60° C.) 30 sec Elongation 60° C.  4 min

After the cycle sequencing purification by precipitation follows:

-   -   ad 2 μl 100 mM EDTA, 2 μl 5M NaOAc (pH 4.8), 1 μl Glycogen        vortex    -   ad von 60 μl 95% ethanol, vortex    -   centrifugation at 13000 g (10 min)    -   remove supernatant    -   wash pellet with 180 μl 70% Ethanol    -   dry pellet    -   resolve pellet in 35 μl Sample Loading Solution (SLS)

Then the probes are pipetted on a microtiter plate overlaid with a dropof paraffin-oil. Then separation in the sequencer at 4.2 V for 60-120min follows. The raw data is analyzed and the sequences are aligned withcontrol sequences using the CEQ 2000 XL software (Version 4.3.9, BeckmanCoulter). Differences between the control sequences (preferably thegenomic VKORC1 nucleic acid sequence or its coding sequence according toSEQ ID NO. 2) and the sequenced DNA is indicative of the probes sequenceto represent a VKORC1 nucleic acid containing a sequence abnormality.

Example 9 PCR-Based Assay for Determining Warfarin Resistance in Rats

In order to determine whether or not a rat (Rattus norvegicus) iswarfarin resistant, i.e. whether the VKORC1 coding sequence according toSEQ ID No. 13 carries a mutation Y139C (416A>G), the following assaybased on ARMS-PCR was employed using rat feces as a source of ratgenomic DNA.

It is the principle of the assay to include into the PCR reaction (1)one PCR primer (rVKORC1-innerF) that specifically hybridizes to the DNAsequence which contains the warfarin resistant mutated allele 416G and(2) another PCR primer (rVKORC1-innerR) which specifically hybridizes tothe Wildtype DNA sequence which contains the Wildtype allele 416A.Moreover, these two primers are oriented in opposite direction such thatthey pair with one out of two additional PCR primers included into thereaction. The latter primers are located in different distances to andin opposite direction relative to the 416 site and as a result,depending on whether the 416 site is mutated or not either therVKORC1-innerR primer or the rVKORC1-innerF primer will anneal and thePCR reaction will result in amplified DNA of a different size which isindicative of the genotype of the rat which DNA has been analyzed. InWildtype rats the PCR reaction will result in a band of 123 bp, whereasin rats homozygous to the mutation 416G the PCR reaction will yield aband at 101 bp. Finally in rats with a heterozygous genotype, the PCRreaction will give rise to two bands, one at 101 and another band at 123bp.

The genomic DNA was isolated from the feces using standard DNA isolationprocedures generally known to the skilled artisan. The followingcomponents were combined to a PCR reaction: 1 μl DNA (rat), 1 μl 5MBetain (Sigma), 2 pmol outer-Primer-F (1 μl of a 1:50-dilution), 2 pmolouter-Primer-R (1 μl of a 1:50-dilution), 10 pmol inner-Primer-F (1 μlof a 1:10-dilution), 10 pmol inner-Primer-R (1 μl of a 1:10-dilution),0.25 μl Taq/Pfu-Polymerase (1.25 U Taq (Invitrogen) and 0.25 U Pfu(Stratagene)), ad 25 μl PCR-buffer (1 ml PCR-buffer contains: 100 μl10×PCR-buffer (Invitrogen), 160 μl nucleotide stem-solution (1.25 mMdNTPs), 30 μl MgCl₂, 610 μl aqua dest). The PCR conditions were: 95° C.for 3 min, followed by 32 cycles of: 95° C. for 20 sec, 62° C. for 20sec, and 70° C. for 10 sec. Finally, the reaction is incubated at 70° C.for 3 min. The PCR Products were separated by gel electrophoresis on a3.5% TAE-Agarose-Gel with ethidium bromide (10 μl of a 1%-stem solutionfor every 100 ml). The gelelectrophosesis was allowed to run for 30 minat 130 V.

The following primers were used:

rVKORC1-outerF: (SEQ ID No. 88) ATC CTG AGT TCC CTG GTG TCT GTC GCT GrVKORC1-outerR: (SEQ ID No. 89) TCA GGG CTT TTT GAC CTT GTG TTC TGG C416-mutant allele-specific PCR primer “rVKORC1-innerF”: (SEQ ID No. 90)TGA TTT CTG CAT TGT TTG CAT CAC CAC ATG416A-wildtype allele-specific PCR primer “rVKORC1-innerR”:(SEQ ID No. 91) CAA CAT CAG GCC CGC ATT GAT GGA AT

Rats (Rattus norvegicus) with and without warfarin resistance were usedfor the assay. The results of the PCR are shown in FIG. 13. Wildtyperats exhibited a band at 123 bp, rats homozygous to the mutationexhibited a band at 101 bp and finally, rats with the heterozygousmutation showed two bands, one at 101 and another band at 123 bp.

As a result, this data demonstrate, that this assay can be employed todetermine whether a given rat is warfarin resistant or not. Such assaysare highly versatile in order to manage pest control in a given region,since knowledge of the frequency of warfarin resistant rats is criticalfor deciding which pesticide may be employed effectively. If in a givenregion there is a high prevalence of warfarin resistant rats warfarinand analogues thereof are an unsuitable means to kill the rats. If,however, the determined frequency of warfarin resistant rats is low,warfarin may be effectively used to fight rodents.

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1. A vitamin K epoxide recycling polypeptide (VKORC1) as a target forcoumarin and its derivatives in mammals comprising a polypeptidesequence selected from the group consisting of: (a) a polypeptidesequence selected from the group consisting of a sequence according toSEQ ID No. 1, 12, and 17; (b) a polypeptide sequence of an allele of thepolypeptide sequence defined in (a); (c) a polypeptide sequence havingat least 80% homology with the polypeptide sequence defined in (a) or(b), wherein the polypeptide sequence has VKORC1 activity; and (d) apolypeptide sequence of a fragment of the polypeptide sequence definedin (a), (b) or (c) having VKORC1 activity.
 2. A VKORC1 nucleic acidcomprising a nucleic acid sequence selected from the group consistingof: (a) a nucleic acid sequence coding for the VKORC1 polypeptide ofclaim 1; (b) a nucleic acid sequence selected from the group consistingof a sequence according to SEQ ID No. 2, 13, and 18; (c) a nucleic acidsequence which hybridizes under stringent conditions to the nucleic acidsequence defined in (a) or (b), wherein the nucleic acid sequence codesfor a polypeptide having VKORC1 activity; (d) a nucleic acid sequencewhich, but for the degeneracy of the genetic code, would hybridize tothe nucleic acid defined in (a), (b) or (c), and wherein the nucleicacid sequence codes for a polypeptide having VKORC1 activity; and (e) afragment of the nucleic acid sequence defined in (a), (b), (c) or (d),wherein the fragment codes for a polypeptide having VKORC1 activity. 3.A fusion protein comprising (a) the VKORC1 polypeptide of claim 1 or apolypeptide encoded by the VKORC1 nucleic acid of claim 2, and (b) aheterologeous part.
 4. A vector comprising the VKORC1 nucleic acid ofclaim
 2. 5. The vector of claim 4, wherein the vector is an expressionvector.
 6. The vector of claim 4, wherein the vector is a knock-out geneconstruct.
 7. A host cell containing the VKORC1 nucleic acid of claim 2.8. The host cell of claim 7, wherein the host cell is a non-humanembryonic stem cell.
 9. A transgenic non-human mammal, wherein thetransgenic mammal contains the host cell of claim
 8. 10. A DNA or a RNAprobe directed against the VKORC1 nucleic acid of claim
 2. 11. A PCRprimer directed against the VKORC1 nucleic acid of claim 2, preferably aPCR primer selected from the group consisting of a PCR primer accordingto SEQ ID No. 53 to 69, and
 70. 12. A small interfering RNA molecule(siRNA) or a short hairpin RNA (shRNA) directed against the VKORC1nucleic acid of claim 2, preferably a siRNA selected from the groupconsisting of SEQ ID No. 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, and50.
 13. An antisense RNA or DNA directed against the VKORC1 nucleic acidof claim
 2. 14. An RNA-aptamere directed against the VKORC1 polypeptideof claim 1, wherein the RNA-aptamere exerts an effect on the activity ofthe VKORC1 polypeptide.
 15. An antibody or a fragment thereof, whichspecifically recognizes and binds the VKORC1 polypeptide of claim
 1. 16.A method of producing a VKORC1 polypeptide comprising the steps of: (I)providing a host cell having been introduced the VKORC1 nucleic acid ofclaim 2; (II) expressing the VKORC1 polypeptide in the host cell; and(III) isolating the VKORC1 polypeptide from the host cell.
 17. A methodof identifying a coumarin derivative which exerts an effect onto theactivity of VKORC1 polypeptide of claim 1 comprising the steps of: (I)providing a host cell having been introduced the VKORC1 nucleic acid ora vector containing the VKORC1 nucleic acid; (II) expressing the VKORC1polypeptide in the host cell; (III) administering a candidate coumarinderivative; (IV) determining the activity of VKORC1 polypeptide(candidate activity value); (V) comparing the candidate activity valuewith a control activity value; and (VI) identifying the candidatecoumarin derivative as a coumarin derivative exerting an effect onto theactivity of the VKORC1 polypeptide, provided the candidate activityvalue is significantly different from the control activity value. 18.The method of claim 17, wherein the control activity value is determinedby a method comprising the steps of: (A) providing a host cell accordingto step (I); (B) expressing the VKORC1 polypeptide in the host cell; and(C) determining the activity of VKORC1 polypeptide (control activityvalue).
 19. The method of claim 17, wherein the determined activity ofVKORC1 polypeptide is dithiothreitol-dependent conversion of vitamin K2,3-epoxide to vitamin K quinone and wherein the significantly differentactivity value is a candidate activity value which is significantlyhigher than the control activity value.
 20. The method of claim 17,wherein at least one additional compound is introduced into the hostcell, which compound is selected from the group consisting of vitamin K,cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase,for microsomal epoxidehydrolase, for calumenin, or forglutathion-5-transferase.
 21. A method of determining a VKORC1polypeptide sequence which conveys a coumarin effect exerted onto VKORC1activity, comprising the steps of: (I) providing a cell expressing theVKORC1 polypeptide of claim 1, which VKORC1 polypeptide has at least onesequence abnormality; (II) administering coumarin or a derivativethereof to the cell; (III) determining the activity of the VKORC1polypeptide (sequence abnormality activity value); and (IV) comparingthe sequence abnormality activity value with the control sequenceactivity value, wherein a significant deviation of the sequenceabnormality activity value from the control sequence activity value isindicative that the sequence abnormality of the VKORC1 polypeptideconveys the coumarin effect exerted onto VKORC1 polypeptide.
 22. Themethod of claim 21, wherein the control sequence activity value isdetermined by a method comprising the steps of: (I) providing a cellexpressing the VKORC1 polypeptide of claim 1; (II) administeringcoumarin or a derivative thereof to the cell; (III) determining theactivity of the VKORC1 polypeptide (control sequence activity value).23. The method of determining of claim 21, wherein the determinedactivity is dithiothreitol-dependent conversion of vitamin K 2,3-epoxideto vitamin K quinone and wherein the significantly different value is asequence abnormality activity value which is significantly higher thanthe control sequence activity value.
 24. The method of claim 21, whereinat least one additional compound is introduced into the cell whichcompound is selected from the group consisting of vitamin K, cytochromeB5, and a nucleic acid coding for gamma-glutamyl-carboxylase, formicrosomal epoxidehydrolase, for calumenin, or forglutathion-S-transferase.
 25. The VKORC1 polypeptide of claim 1, whereinthe VKORC1 polypeptide contains at least one sequence abnormality, whichexerts an effect on the activity of the VKORC1 polypeptide.
 26. TheVKORC1 polypeptide of claim 25, wherein the VKORC1 polypeptide is thepolypeptide according to SEQ ID No. 1 or 12 and the sequence abnormalityis selected from the group consisting of V29L, V45A, R58G, R98W, L128R,and Y139C.
 27. A VKORC1 nucleic acid selected from the group consistingof: (a) a nucleic acid coding for the VKORC1 polypeptide of claim 25 or26, (b) a nucleic acid sequence selected from the group consisting of asequence according to SEQ ID No. 3, 4, 5, 6, 7, 14, and 94, (c) anucleic acid sequence which, but for the degeneracy of the genetic code,would hybridize to the nucleic acid defined in (a) or (b), and whereinthe nucleic acid sequence codes for the polypeptide of claim 25 or 26.28. A vector containing the VKORC1 nucleic acid of claim
 27. 29. A DNAor a RNA probe directed against the VKORC1 nucleic acid of claim
 27. 30.A PCR primer directed against the VKORC1 nucleic acid of claim
 27. 31.An antibody or a fragment thereof, which specifically recognizes andbinds a VKORC1 polypeptide of claim 25 or
 26. 32. A transgenic non-humanmammal wherein the mammal contains a stem cell containing the VKORC1nucleic acid of claim 27 or the vector according to claim
 28. 33. Adiagnostic comprising a compound selected from the group consisting of aVKORC1 nucleic acid of claim 27, a DNA or the RNA probe directed againstthe VKORC1 nucleic acid of claim 27, a PCR primer directed against theVKORC1 nucleic acid of claim 27, and an antibody or a fragment thereof,which specifically recognizes and binds the VKORC1 polypeptide (VKORC1)as a target for coumarin and its derivatives in mammals comprising apolypeptide sequence selected from the group consisting of: (a) apolypeptide sequence selected from the group consisting of a sequenceaccording to SEQ ID No. 1, 12, and 17; (b) a polypeptide sequence of anallele of the polypeptide sequence defined in (a); (c) a polypeptidesequence having at least 80% homology with the polypeptide sequencedefined in (a) or (b), wherein the polypeptide sequence has VKORC1activity; and (d) a polypeptide sequence of a fragment of thepolypeptide sequence defined in (a), (b) or (c) having VKORC1 activity;wherein the VKORC1 polypeptide contains at least one sequenceabnormality, which exerts an effect on the activity of the VKORC1polypeptide; or wherein the VKORC1 polypeptide contains at least onesequence abnormality, which exerts an effect on the activity of theVKORC1 polypeptide; and wherein the VKORC1 polypeptide is thepolypeptide according to SEQ ID No. 1 or 12 and the sequence abnormalityis selected from the group consisting of V29L, V45A, R58G, R98W, L128R,and Y139C.
 34. A method of diagnosing a VKORC1 associated deficiency ina patient comprising the steps of: (I) amplifying a DNA sample obtainedfrom the patient or reverse transcribing a RNA sample obtained from thepatient into a DNA and amplifying the DNA; and (II) analyzing theamplified DNA of step (I) to determine at least one sequence abnormalityin a nucleic acid sequence coding for the VKORC1 polypeptide of claim 1or in an amino acid sequence of the VKORC1 polypeptide; wherein thedetermined sequence abnormality is indicative of the patient sufferingfrom a VKORC1 associated deficiency; preferably warfarin resistance. 35.The method of claim 34, wherein the amplified DNA encodes at least apartial sequence of the VKORC1 polypeptide according to SEQ ID No. 1 andwherein the sequence abnormality is selected from the group consistingof V29L, V45A, R58G, R98W, L128R, and Y139C.
 36. The method of claim 34,wherein the amplified DNA is analyzed by a technique selected from thegroup consisting of PCR-based analysis, restriction digestion analysis,and DNA sequencing analysis.
 37. A method of diagnosing a VKORC1associated deficiency in a patient comprising the steps of: (I)providing a sample obtained from the patient; and (II) detecting aVKORC1 polypeptide having a sequence abnormality in the sample using theantibody of claim 31, wherein the determined sequence abnormality isindicative of the patient suffering from a VKORC1 associated deficiency.38. The method of claim 37, wherein the sample is analyzed by atechnique selected from the group consisting of immunohistochemicaldetection, immunoblotting, preferably Western blotting, and ELISA.
 39. Amethod of identifying a coumarin derivative which exerts an inhibitoryeffect onto the activity of a VKORC1 polypeptide having at least onesequence abnormality, comprising the steps of: (I) providing a cellexpressing the VKORC1 polypeptide according to claim 25 or 26,preferably a polypeptide encoded by a sequence selected from the groupconsisting of SEQ ID No. 3, 4, 5, 6, 7, 14, and 94; (II) administering acandidate coumarin derivative to the cell; (III) determining theactivity of the VKORC1 polypeptide (sequence abnormality activityvalue); and (IV) comparing the sequence abnormality activity value withthe control sequence activity value, (V) identifying the candidatecoumarin derivative as the coumarin derivative exerting an inhibitoryeffect onto the activity of a VKORC1 polypeptide, if the administrationof the candidate coumarin derivative results in a sequence abnormalityactivity value which is significantly lower than the control sequenceactivity value.
 40. A method of identifying a coumarin derivative whichis toxicologically effective in warfarin-resistant rodents comprisingthe steps of: (I) providing a warfarin-resistant rodent; (II)administering a candidate coumarin derivative to the rodent; (III)determining the toxicity of the candidate coumarin derivative onto therodent (candidate coumarin derivative toxicity value); (IV) comparingthe candidate coumarin derivative toxicity value with a control coumarintoxicity value; (V) identifying the candidate coumarin derivative as atoxicologically effective coumarin derivative, provided that thecandidate coumarin derivative toxicity value is significantly largerthan the control coumarin toxicity value.
 41. A method of identifying acoumarin derivative which is toxicologically effective inwarfarin-resistant rodents comprising the steps of: (I) providing awarfarin-resistant rodent; (II) administering a candidate coumarinderivative to the rodent; (III) determining the toxicity of thecandidate coumarin derivative onto the rodent (candidate coumarinderivative toxicity value); (IV) comparing the candidate coumarinderivative toxicity value with a control coumarin toxicity value; (V)identifying the candidate coumarin derivative as a toxicologicallyeffective coumarin derivative, provided that the candidate coumarinderivative toxicity value is significantly larger than the controlcoumarin toxicity value; wherein the warfarin-resistant rodent is arodent transgenic for VKORC1 polypeptide of claim 1, wherein the VKORC1polypeptide contains at least one sequence abnormality, which causeswarfarin resistance, preferably a polypeptide encoded by the sequenceaccording to SEQ ID No.
 14. 42. The method of claim 40, wherein VKORC1polypeptide is the polypeptide according to SEQ ID No. 12 and thesequence abnormality is selected from the group consisting of V29L (85G>T), V45A (134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383T>G), and Y139C (416 A>G).).
 43. A composition for killing rodents,comprising a rodenticidally effective amount of the coumarin derivativesidentified by the method according to claim
 39. 44. Use of the PCRprimers according to SEQ ID No. 88 to 91 for determining whether or nota rat has a warfarin resistance genotype in a sample obtained from arat.
 45. The vector of claim 5, wherein the vector is a knock-out geneconstruct.
 46. A host cell containing the vector of any one of claims 4through 6 and
 45. 47. The host cell of claim 46, wherein the host cellis a non-human embryonic stem cell.
 48. A transgenic non-human mammal,wherein the transgenic mammal contains the host cell of claim
 47. 49. Amethod of producing a VKORC1 polypeptide comprising the steps of: (I)providing a host cell having been introduced a vector containing theVKORC1 nucleic acid of any one of claims 4 through 6 and 46; (II)expressing the VKORC1 polypeptide in the host cell; and (III) isolatingthe VKORC1 polypeptide from the host cell.
 50. A composition for killingrodents, comprising a rodenticidally effective amount of the coumarinderivatives identified by the method according to claim
 40. 51. Acomposition for killing rodents, comprising a rodenticidally effectiveamount of the coumarin derivatives identified by the method according toclaim 41.